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Full text of "Inhibition of human complement by extracellular lipoteichoic acid from Streptococcus Mutans BHT"

INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLULAR 
LIPOTEICHOIC ACID FROM STREPTOCOCCUS MUTANS BHT 



By 

LOUIS JOSEPH SILVESTRT 



A DISSERTATION PRESENTED TO Till'. 
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA 
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DECREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1 1) 7 7 



ACKNOWLEDGEMENTS 

In all sincerity, no occasion or project thus far undertaken has had 
a more humbling effect on my life than the completion of this Ph.D. disser- 
tation. It is unfortunate that only now in retrospect can I clearly see the 
tremendous debt I owe for the help, patience, understanding, and knowledge 
so generously contributed by my mentor, my friends and wife. 

If I were to formally thank everyone who contributed in someway to the 
successful completion of my Ph.D. dissertation, it is likely that my acknow- 
ledgements would read like the listings of a telephone hook. I do however 
feel compelled to thank a few very special people. 

First of all I would like to thank my mentor and friend, Dr. E.N. 
Hoffmann. Ed possesses the rare ability to earn respect rather than having 
to demand it. His tolerance for my id i osvncrasies was unlimited (almost). 
He gave me direction yet left me with alternatives; he challenged my intellect, 
yet never made me feel ignorant; he provided the foundation on which 1 am 
still building my scientific character. Most of all, he was (and is) a friend 

I would also like to thank the members of our laboratory "family" 
(Suzanne, Jean, Bert and Tom) for their help, patience and tolerance 
during these difficult last days. One cannot help hut reflect upon the many 
events that shape the complex web of friendships within the laboratory. 
All of you will always he regarded as the closest of friends. 

I wish to express my gratitude to Ron Craig not only for his friend- 
ship, but also for the professional technical, assistance that he afforded. 

1 would also like to express my appreciation to the employees of Teach- 
ing Resources, for dedication above and beyond the call of duty. I would 



i i 



especially like to acknowledge the professional artistic assistance o r 
Margie Summers, Margie Niblack and John Knauh. 

I would like to thank Steve Hurst for being there when I needed a 
friend and for helping with some of the last minute photography. 

The typist, Joanne Hall, deserves a particularly special mention. 
If not for her personal concern and dedication the deadlines would never 
have been. met. She worked on this dissertation under conditions for which 
no degree of monetary reinbursement could possibly compensate. T thank 
you Joanne and I sincerely hope you never have to go through that again! 

Finally, and most importantly, I wish to thank my wife Lvn for her 
infinite patience and encouragement. Her attitudes, her ideals, her 
'being" is so much a part of me that it would lie hopelessly futile to 
list all the things for which 1 am indebted to her. She is a friend 
and lover, a typist and an occasional laboratory technician. She is rsv 
driving force in life and rightfully so, I dedicate this dissertation 
to her. 



And i\),i,th one load iuoilcuvo'"i'-iatvo'ii<u>jcn>iai< ] r'i'ia he. jumped at the 
and of, tkt tablecloth, pulled it to the gxound, wrapped himeCf, up in i 
tJviee. times, 'tolled to the. otlici end o& the loom, and afitei a teitibtc 
ithuggle, got hi,\ head into datjiigiit aaa in and said chcci ;\nCCu 
-- have \ mm?" 

U*[pm "The House at fVc/i Cc'atei" bn A. A. MiCne.) 



TABLE OF CONTENTS 

PAGE 

ACKNOWLEDGMENTS i i 

LIST OF TABLES v 

LIST OF FIGURES vi 

GLOSSARY OF ABBREVIATIONS viil 

ABSTRACT ix 

INTRODUCTION I 

MATERIALS AND METHODS 1. 7 

RESULTS 11 

DISCUSSION 1 OS 

LITERATURE CITED 1 20 

BIOGRAPHICAL SKETCH 132 



list: of tarf.es 



TABLE 
1. 



5. 



7. 



10. 



11. 



Partial Purification of LTA by A5-M 

Gel Filtration 

I. Results from Partial Purification 

of LTA 

II. Results from Partial Purification 

of LTA 

Percent Recovery of LTA During Octyl 
Sepharose Purification 

Distribution of C-Phosphat idvl Choline 
During PCV Puri f ication of LTA 

Percent: Recovery of LTA from Various 

Steps of PCV Purification , 

Summarized Chemical Composition of Various 
LTA Containing Sources 

Specific Activity Determinations of Purified 
LTA 

Effect of LTApcx on the Anility of Cls fn 
Consume ('.'> and C2 Activity 

Comparison of tire Relative Numbers o\~ 
Effective CI Molecules Capable of Transfer 
from FAC1 Treated with LTApcx 

The inhibition of FA lysis by Li potof choir 
Acids from Several Bacterial Sources 



PACE 



57 



hi 



63 



73 



70 



1(17 



ST OF FIGURES 



FIGURE 
1. 

2. 



6. 



7. 



8. 



10. 



11, 



12. 



Titration of whole human complement 
after incubation with crude extra- 
cellular Lipoteichoic acid (LTAcx) 

Hose response inhibition (if whole 
human complement after incubation 
with varying concentrations of LTAcx... 

Titration of C'3 in whole human serum 
after treatment with LTAcx 

Complement component titration of 

whole human sera after treatment 

with LTAcx 

Inhibition of complement mediated 
lysis of FA treated with varying 
concentrat Lons of LTAcx 

Passive hemagglutination (TllA) of F.A 

treated with varying concentrations 

of LTAcx 

Effects of LTAcx treatment on the 
lysis of various complement component 
intermediates 

PI1A of various LTAcx treated complement 
romp on ant in termed Latcs 

Effect of LTAcx treatment en tin- lysis 
oi EAC142 

Effect of LTAcx on hemolytic antibody 
t i t rat ion 

Partial purification ot l.TA hv A5-M 

Re 1 f i 1 t ra t ion 

Partial purification of LTA by A'j-M 

gel filtration with I TA enriched 

start In g mater In I 



PAG I 



33 



35 



38 



43 



48 



30 



r i9 



h] 



v \ 



13. Purification of I.TA by Oc.tyl Spphnrose 
hydrophobic go I chromatography 

14. Simultaneous removal of salt and propanol 

from LTAosx by Ul-20 gel chromatography &9 

15. Carbohydrate analysis of f.TA containing 
preparations by gas liquid chromatography "6 

16. Passive hemagglutination (PHA) titration 
and inhibition of complement mediated lysis 
of EA treated with varying concentrations 
o f LTApcx • 



17. Effect of LTApcx on the complement 
mediated lysis of various cellular 
complement component intermediates 

18. Effect of LTAcx and LTApcx on function- 
ally purified human CI. 

19. Immunodiffusion and precipitation analysis 
of various steps in the purification of 
human Clq • ■ 

20. Disc gel electrophoresis of purified 
human Clq 

21. DEAE elution profile of human Cls 

22. Immunoelectrophoresis of human Cls and 

cls 

23. Effect of LTApcx on the ability of Cls 

to hydrol i ze TAMe 



Difference in complement mediated ly tie 

susce 

EAC14 



susceptability of LTApcx treated EAC4 versus 



81 



8 3 



8ft 



88 



•>n 



c n 



9° 



(13 



vi i 



GLOSSARY OF ABBREVIATIONS 



A: 




Antibody 


C: 




Complement 


CI, 


C2-- 


-- C9: a Com 



Complement components. Horizontal bars 
above the component designation denotes 

a biologically active state. 

CVF: Cobra venom factor 

E: Erythrocyte 

EDTA: (Disodium) lit by lened i amine tetraaeetic acid 

EGTA: Ethy lenegj ycol-b is (6 Amino Ethyl Ether) N,N- tetra 
acetic acid 

LTAcx: Crude extracellular iipote.ichoic acid 

LTApcx: Extracellular LTA purified via phosphatidyl-choline 
vesicle adsorbt: ion 

LTAppx: Partially purified extracellular LTA 

LTAosx: Extracellular LTA purified via Octyl Sepbarose gel 
column adsorbt ion 

LPS : L ipopolysaceha r i de 

PHA: Passive hemngglut i nation 

PHAg: Passive hemagglutination (modified method) 

TA: Teichoic ac i d 

TAME: p-Tosy 1 - 1 -nrg i n i tie methyl ester 



a 

All complement nomtnu: 1 a ture follows the WHO recommendation.' 

(Bull. Wld. Hlth. Org. 39:439, 1968). 



v i i i . 



Abstract of Dissertation Presented to the 

Graduate Council of the University of Florida 

in Partial Fulfillment of the Requirements for the 

Degree of Doctor of Philosophy 



INHIBITION OF HUMAN COMPLEMENT BY EXTRACELLULAR 
LIPOTEICHOIC ACID FROM STR EPTOC OCCUS HUT AN S BHT 

By 

Louis Joseph Silvestri 

December, 1977 



Chairman: Edward M. Hoffmann 

Major Department: Microbiology and Cell Science 



A number of biological and chemical similarities exist between 
the lipopolysaccharides (LPS) of pram negative microorganisms and the 
lipoteichoic acids (LTA,) of gram positive organisms. The potent 
affects of LPS on the complement system are well documented; however, 
the effect of LTA on this host defense system has not been adequately 
studied. Furthermore, all studies thus far conducted have been limited 
to the interaction of LTA with whole fluid phase complement. In this 
investigation it was demonstrated that extracellular LTA from the 
cariogenic microorganism Streptococcus mutnns HUT was not only capable 
of spontaneously binding to sheep erythrocyte target cells but was 
also capable of rendering them refractory to complement mediated lysis. 
Purification of the LTA to homogeneity was achieved by a combination of 
gel filtration and adsorbtlon to phospholipid choline vesicles (arti- 
ficial membranes). By utilizing various cellular complement component 
intermediate complexes and functional ly purified complement components, 
experiments were conducted to define the site and mechanism of Inhibition 



i x 



by LTA. The site of Inhibition was determined Co occur between the 
formation of the SAcT and SAcT42 complex. Because Ci is no longer 
necessary after formation of the C3 convertase (SAC42), lack of inhi- 
bition after this step implies a direct effect on Ci activity. Although 
experimental data derived from utilizing CI, Clq , Cls, and Cls were 
suggestive, data did not unequivocally establish this as the precise 
mechanism of inhibition. No evidence for fluid phase consumption of 
hemolysin Ah, CI, C4, or C2 by LTA could be demonstrated. Evidence for 
the inhibitory activity of LTA from several unrelated genera is pre- 
sented and the possible role of LTA in periodontal, disease is discu^-1. 



INTRODUCTION 



As reviewed by Wicke.n and Knox (1,2), n number of chemical and 
biological similarities exist between the 1 ipopo Lysnccharides (LPS) of 
gram negative bacteria and the lipoteicboie acids (LTA) of gran positive 
organisms. Because of these similitudes our Laboratory began to inves- 
tigate whether LTA possessed ant t complementary activity analogous to 
that associated with the LPS endotoxin (3-8). Although there have been 
concentrated efforts to define the site and mechanism of LPS inhibition of 
complement, very few investigators have reported data on the possible 
effects of LTA on the complement system (9). This is somewhat surprising 
since the interaction of LTA, LPS, and complement almost certninlv plav a 
significant role in the etiology of periodontal diseases. Bacterial pro- 
ducts and serum components in (Tie gingival crevices of the oral cavitv 
have been shown to activate complement by both the classical (10,11) and 
the alternative pathways (12,13). In fact recent: evidence suggests that 
bone loss (a major clinical manifestation of acute periodontal disease) 
may occur via osteoclast activation due to the interaction of complement 
and prostaglandin E (!'*)• Prostaglandins are naturally occurring eyclized 
derivatives of unsaturated long chain fatty acids (15) and their concen- 
trations are dramatically elevated in inflamed gingival tissues (16). Tt 
is of interest to note, that both LTA and LPS are also capable of initiating 
osteoclast mediated bone resorption (171 and this activity proceeds with- 
out the contribution of romp 1-ement or prostaglandins. The potential I'^v 
synergism cannot be overlooked, and indeed LPS endotoxins have lone hoen 
implicated as participants in the development of periodontal legions (4-fl). 
Analogous LTA activity could lie of significant clinical import especially 



in light of the fact that gram positive bacteria represent the major 
cellular constituent of dental plaque at the early stages of plaque 
formation (18). Most of the gram positive organisms found in dental 
plaque have been Isolated, cultured, and identified. The production of 
copious amounts of extracellular LTA by several of these organisms has 
been well established (19,20). In fact, growing under conditions esti- 
mated to reflect the growth rate in the oral cavity, Wicken and Knox have 
shown that the cariogenic bacterium Strepto coccus rmitans BHT produces 
some eleven fold greater amount of extracellular LTA in the culture fluid 
than that contained within the cells themselves (1,2). Therefore, if an 
effect on complement by LTA can be demonstrated til vitro, an in vivo 
model can be readily envisioned. Preliminary experimentation with n 
crude LTA containing extract from S. mu tans RUT did indeed indicate that 
complement activity was consumed. However, consumption or alteration 
of complement activity can be due to a number of specific or non-specific 
factors. Because of the complexity of this system, a thorough under- 
standing of the possible interactions is necessary before anv model 
attempting to define a site and mechanism of inhibition can be elucidated 

The complement system of vertebrates is comprised of at least 
eighteen discrete plasma proteins capable of interacting in a specific 
and sequential fashion. There are two pathways by which this biochem- 
ical cascade may bo initiated and they are referred to as the classical 
and the alternative pathways of complement activation. However, regard- 
less of bow the activation scheme is initiated, the biological consequen- 
ces of activation are the same for both pathways: 



1 



Silvestri et al. 1976. Abst. Ann. Meet ins:, ASM, p77 



3 



1.). phLogogenic activity mediated via complement reaction by-products 
2). increased opsonic susceptibility of foreign substances 
3). irreversible phys iochemicaj membrane damage— and ultimately, cvto- 
Lysis — of susceptable target coll;;. Although the importance of comple- 
ment as a component of the host defense system has been suspected for 
quite some time, only recently has its biomedical significance been 
firmly established. Indeed, the participation of complement in host 
resistance to infections and In several disease mechanisms is a topic 
which has generated considerable research interest: in recent years (21,22) 

The classical pathway of complement contains eleven discrete glyco- 
proteins representing nine distinct components referred to sequentially 
as CI through C9. CI is actually a multimelecu.lnr complex of three dis- 
tinct proteins (Clq, Clr, and Cls) and the aggregate is hold together hv 
the divalent calcium ions (23). Removal of calcium inns by chelating 
agents such as othy Iened iani i ne tetraacct i c acid (EDTA) results in the dls- 
association of CI into its subcomponents with concomitant loss of activity 
(24). Activation of the classical pathway i? characterized by a depen- 
dence on IgG or 1 gM antibodies complexed with antigens. The classical 
pathway also specifically requires the components CI, C2, and C4 as we 1 1 
as the divalent cations calcium and magnesium, Although the components 
C3, and C5 through C9 are usually considered part of the classical system, 
they are shared by the alternative pathway and thus are nut considered as 
unique components of the classical system per se . 

The recognition and hill i. it ion fund Ion with respect to Immuno- 
globulins resides with the Clq subcomponent (2*5,26). Clq itself is a 
rather peculiar protein consisting of three different" polypeptide chains 
(27). Chemically, Ctq contains approximately 10"' carbohydrate. 3'' 



hydroxyprol ine , 27, hydroxy lysine and 13" glycine. This unusual collngen- 
like composition makes i.t unlike any plasma protein yet described (28,29). 

When complement is activated by ant ihody-antigen complexes such as 
exists on the surface of an antibody sensitized erythrocyte (EA) , it 
undergoes a seJ f assembly process sequentially depositing the entire 
fluid phase, cascade onto the surface of the target. Specifically, Ciq 
recognizes a previously sequestered binding site located in the Fc frag- 
ment of IgG and IgM (30,31). The three polypeptide chains of Clq are 
physically arranged in a manner perhaps analagous to a six headed mace 
or bola with each "head" representing a binding site for an IgG molecule 
(32). Thus each Clq molecule has six binding sites for IgG (and presum- 
ably the same number for IgM). Internal activation of CI probably is the 
result of a conformational change in Clq which in turn induces a change 
in the proenzvme Clr (33). Once Cl.r is activated to Clt it is endowed 
with enzymatic activity through which the proenzyme Cls is converted to 
CI esterase (cis )(3-'s 35, 3M . ('Is is a serine esterase and is inhibited 
by diisoprnpy Iphosphof luor idate (DFP) (37). This esterase activitv can 
be used to hvdrolyze the synthetic substrates p-Tosv 1 - I argin ine methv- 
.1 ester (TAMe) and N-ac.ety 1-1-tyrosine ethytester (ATi'e) (38). Recently. 
Loos and Raepple have demonstrated that many polyanions were capable of 
inhibiting the activity of CI either hv interfering with Clq binding to 
the antibody-antigen complex, or by preventing interaction ol CA and C 1 
with cTs (39, 40). Although binding of CI usually leads to activation, 
the two processes are net integral— Ig^ with modified tryptophan (41) 
and the human immunog 1 obti 1 i n subclass IgG* (62) — both bind Clq but do 
not act i va t e CI. 

After activation, Cls en:-'.ymat ica 1 ly cleaves C't into a large (C4b) 



and small fragment (C4a) (A3). The cleavage of C4 expose, n rembrane 
attachment site on the C4b molecule and it will attach to the antibody- 
antigen complex at a site juxtaposed to the Ct-antibody complex (44,45). 
cTs then cleaves C2 into C2n and C2b (46) with C2a attaching to the C4h 
site and C2b being released into the fluid phase. Thus, the molecular 
complex C4b2i is formed and is referred to as C3 convcrtase because it 
is capable of splitting and activating C3 (47,48). C3 convertase is 
also an esterase, and although C3 is its natural substrate, it also 
hydrolyzes the ester bond of acetvl-glvcl- lysine methyl ester (49). The 
catalytic site of C3 convertase is believed to reside in the C2a sub- 
unit and even after release from the C4b complex, cvtolv t ical lv inactive 
C2a retains esterase activity, but is no longer capable of cleaving C3 
(49). The enzymatic half-life of cTbla" is quite ephemeral-only 10 
minutes at 37°. However, if the C2 is first oxidized by treatment with 
iodine (applicable to human but not guinea P ig C2) . not only Is the 
binding of C2a to C4b enhanced, but the half life of the binolecular 
complex is increased 20 fold (30). No doubt: the transient association 
of C2a with the C42 and 0423 complex plays a vital role in controlling 
Che complement reaction by temporarily limiting the functional assooia- 
tion of these complex enzymes. 

nceC3 is cleaved intoC3n and C3b, the small C.3a Fragment is 
released into the fluid phase and C3h becomes associated with the C4b2a 

complex and with other non-hemolvMc si.es on the target mc -no (47). 

The association of 03b with the 0,3 convertase modulates it, activity so 
that now fS becomes the natural substrate ot this t r i no I ecu 1 ar complex. 
:4TIb complex is referred to as C5 convertase (31) and like C42, is 



The C4 2 3b 

a highly snecial Ued pretense 



!u ,, t - ;1S C ) i s the only known protein 



substrate for C42, C5 is the only known substrate lor r.423. 

Once C5 is cleaved into C5a and C5b, <'/">a is released in the fluid 
phase and C5b transiently acquires the ability to hind one molecule 
each of C6 and C7 (52,53). With this, a self-assembly process is ini- 
tiated and results, without any further enzymatic activity, in the form- 
ation of the stable C5b-9 complex (54). It should be noted that the 
small by-product fragments C3a and C5a are endowed with marked phlogo- 
genic activity (55,56,57). Some of these activities include, release of 
histamine from mast cells, contraction of smooth muscle tissue, directed 
chemotaxis of polymorphonuclear leukocytes, and vasodilation both in con- 
junction and independent of histamine activity (58). Such potent pharma- 
cological activities obviously play a major role in the normal course 
of the inflammatory response. i 

Once the C5b67 complex is formed, it too can hind nonspec i f ica! 1 v 
to areas on the membrane other than at the location of the C5 eonvertase 
(52). The trLmole.cuiar association of C567 provides the molecular arrange- 
ment for the adsorptive binding of one molecule of ('8 which in turn pro- 
vides a binding region for up to six molecules of C (1 (54). A Low grade 
lesion of the target membrane occurs with only the addition of C3 to th 
complex '(■}«)); but with the binding of C9, a ten component maorcno I ecu ! ur 
eomplex is formed which greatly enhances the rate of target cell evt.v- 
lysis (54). It should h" nofed that the C5hh7 complex or even the C5hh7 
complex can attach to non-sens i t I zed "innocent by-stander" cells and thus 
Promote a terminal cytolytic: event. This phenomenon has boon termed 
"reactive lysis" (60) and is controlled by the rapid de-av of the unbound 

eompl ex C>! ,62) . 

The precise mechanism by which complement mediates cvtelvsis of 



susceptible target cells is not dearly understood. One hvnothesis, in 
light of t'n<.> newly discovered tr ibtityr innso activity of r.7 , is that the 
lytic event is caused by an enzymatic attack on the membrane (63). How- 
ever, no enzymatic degradation products have ever been discovered in 
either lysed cell membranes or in ruptured synthetic lipid hi Lava rs (64). 
The two most favored models are the "doushnut" insertion hypothesis (65) 
and the C8 insertion model (29). The former model purports that the 
C5b-9 complex inserts inself into the membrane as a "prefabricated bole" 
allowing the exchange of intra and extracellular material via an interna! 
hydrophilic channel. (65). However, the model fails to explain hew the 
hydrophil ic complement components enter the hydrophobic expanses of the 
membrane. In addition, although electron microscopy has revealed apparent 
ultrastrueture doughnut shaped "lesions" on the surface of cells I.yse.d by 
complement (66), freeze etching techniques have shown that the ultra- 
structure alterations are confined to the outer leaflet of the membrane, 
i.e. the lesion does not penetrate the membrane (67). The Co Insertion 
model embraces most of the salient features of the doughnut model, but 
in addition postulates that the a and y chains of C3 are inserted Into 
the channel formed by the surface macromo 1 ecu 1 ar complex. The -■ md y 
chains thus extend into the membrane bilaver causing disruption of order Iv 
st rue ture. 

In addition to the restraints placed on the complement cascade duo 
to the rapid decay of several of the intermediates, there are two 
naturally occurring inhibitors of complement present in the sera ol man 
and prolia.li lv in all ver t. ebra t es . the first inhibitor is referred tn )s 
Cls inhibitor and, as the namo implies, it directly abrogates the heme- 
Lytic and esterol.vtie activity of (11 (68,69). The second inhibitor is 



referred to as C3b Inactivator and cleaves both soluble and rell bound 
C3b into two antigenicaily distinct fraRraents, C3c and C3d (70). As a 
result, C423 loses C5 convertase activity, and C3b activation of both 
the alternative pathway and the immune adherence phenomenon is abolished 
(71,72,73). This latter activity can be visualized by the clustering of 
cells bearing C3b on their surface around other colls displaying C3b 
receptors. Such receptors have been shown to be present on human erythro- 
cytes , polymorphonuclear leukocytes, platelets, macrophages, and on I 
lymphocytes (74,75). The attachment of C3b not only plays a direct role 
in the increased opsonization of target cells (76), but C3b binding to B 
lymphocytes has been postulated to play a role in B-ceJ I activation as 
well (77). 

The second pathway by which complement may be activated is referred 
to as the alternative or properdin pathway. Historically, the existence 
of this pathway had been suggested as early as 19 r >A. At that time, 
Pillemer and his associates reported the discovery of a new protein in 
normal human sera (78). Properdin, as it was called, was capable of 
reacting nnn-specif ically with diverse naturally occurring polysaccharides 
and I ipopo 1 ysaceharides ultimately resulting in the activation ^f comple- 
ment. This process ostensibly occurred without the interaction of anti- 
body and was proposed as a major pathway hv which susceptible bacteria 
and viruses were destroyed. However, this provocative hypothesis was 
discarded as apocryphal and the described activities? wore attributed to 
the presence of natural antibodies (79). The controversy remained un- 
resolved until recent years when rigorous immunochemical techniques wore 
employed in the Isolation, purification, and determination of function 
of many of these components. I'he unanticipated complexity of the properdin 



system has spawned a multiplicity of models attempting to elucidate its 
precise made of initiation and function. Clearly, a plethra of diverse 
stimuli are capable of activating this pathway, and this fact alone im- 
poses a formidable constraint on any molecular model. Some of the more 
common naturally occurring activators of the alternative pathway Include 
bacterial and fungal cell wall constituents such as I ipopolvsae-har ide , 
zymosan, and inulin (a po 3 y fructose) (71,80-83). in addition, aggregates 
of some immunoglobulin classes (84,85), some types of animal cell mem- 
brane constituents (86,87), and antibody-coated budding virus infected 
cells (88,89) also stimulate this pathway. The alternative pathway can 
even be activated by substances of relatively defined chemical nature 
such as benzvl-B-D-fructopvranoside (90), nolvglucoso with repltitlous 
a 1-3 and branched a 1-6 linkages (91), d tnit ropheny lated albumin (92), 
and many polyanionic substances. Cobra venom factor (a non-1 ipo lytic, 
non-hemolytic glycoprotein isolated from Live venom of the cobra Naga 
naja) is also a potent activator of complement cytolytic potential, but 
it appears to act as a C3h analog and is thus unique in its mode of 
alternative pathway activation (93,94,95). Potentiation of this system 
requires devalent magnesium ions and the interaction of at least five 
novel serum proteins. By convention, the names of these proteins are IF 
(or initiating factor), P or P (properdin). Factor B (CI pmnct ivntor) , 
Factor B (C3 activator), and Factor 1) or 1) (C3 pronetivalor oonve rtase ) • 
To date, all of the above components have been isolated, purified, and 
characterized as to molecular weight, e! ect rophnretic mobility, and sedi- 
mentation coefficients (83.96-98). C3b (pi the classical pathway) plays 
an intregal role in the alternative pathway (71,96,99). and thus it in 
essence forms the junction point of the two systems. Because all terminal. 



components (C3, C5-9) are shared, the biological consequences of acti- 
vation encompass all the processes previously described (immune adherence, 
opsonic activity, anaphy latoxin production, membrane attack complexes, 
etc.). 

There are similarities between some of the more salient features 
of the classical pathway compared with those of the alternative pathway. 
Analogous to Clq, IF seems to function as the recognition unit for the 
properdin pathway, but its relationship to another factor (referred to 
as a C3 nephritic factor from the sera of patients with membranoprolifera- 
tive glomerulonephritis (100) and its mode of activation is poorly under- 
stood (96). Factor 1) is capable of enzvma t lea 1 1 y cleaving Factor B into 
Ba and lb (29, 94). In the presence of C3h, a bimolecular complex ClblMi 
is formed (29) which is endowed witii C3 splitting activity similar to 
the C3 convertase (C4b2a) of the classical pathway . Furthermore, .just 
as CAb anchors the classical convertase. to the membrane allowing C2a to 
exert its enzymatic activity, so too cytophilic (Fib anchors the C3bRb 
complex to the membrane allowing the enzymatic activity of Factor Bb to 
be expressed (83). Both complexes merely gain additional C3h to modulate 
C5 cleaving activity (99). Thus, the presence of C3 not. onlv prevents an 
"abort" due to rapid decay of either convertase, but because (Mb is 
utilized as part of the alternative pathway convertase, it participates 
in a type of amplification loop. Tn other words, the more C,3h that: is 
formed from either pathway, the more C3 cleaving potential is endowed 
upon the properdin C3 convertase. Properdin (?) seems to stabilize the 
fragile. C3h.Bb complex but its possible role in stabilizing the classical 
''3 convertase has not been investigated (99). NotPworthv, however, is 
the. potent effect properdin exerts en the C3h inhibitor (99). By 



modulating the action o\ this enzyme, properdin at least Ind i* reef ly 
plays a role in stabilizing the classical pathway sequence. 

The recognition of foreign substances by a host usually leads to 
the neutralization and eradication of these substances by immune lympho- 
cytes, phagocytic cells, specific antibodies, complement, or an amalga- 
mation of these factors. However, in instances where antigenic substances 
interact directly with host tissue, the reactions of the host's immuno- 
logical defense system could sometimes result in a considerable amount 
of autodestruc t ion. LTA represents a class of antigens that are capable 
of spontaneous cytophllic binding to mammalian tissue (101,102,103). 
As a result, host tissue acquires a new "anitgenic face" and may now 
react with natural or Induced antibodies to the LTA. Furthermore, anti- 
bodies directed primarily at LTA determinants may cross react with 
similar determinants of the host's tissue. Such a mechanism has been 
proposed for the high incidence of rheumatic fever and glomerulonephritis 
in patients recovering from post streptococcal infections (104,105). 
Recently, acylated heteropo ly saccharides (LI'A) isolated from the cell 
membranes of several Lactobacillus species were shown to replace pigeon 
excreta antigens in complement consumption tests diagnostic for pigeon 
breeders disease (9,106). Thus, precedence mav already be established 
for LTA's role in the manifestation of several clinical maladies. in 
addition, the. chemical and biological similarities between LTA .and LPS 
(1,2) plus the ability of LTA to stimulate bone resorhtion (17) make 
LTA a likely candidate for a role in periodontal disease. On the other 
hand, LTA lacks some ^i' the biological activities associated with LPS 
such as pyrogenioity in rabbits (.2,107) and a mitegonic effect on B-celts 
(2). Since these activities have been shown to reside with the complex 



12 



Lipid A of LPS (.108,109) and since the unique sugars and hvdroxvacy I 



iSters of Lipid A are absent in LTA, it is not 



surprising that asscei.it' 



activities are absent as well. As a class, teichoic and lipoteichoic 
acids are wall and membrane components of gram positive bacteria (107,108) 
LTA is typically membrane associated and consists of a glycolipid cova- 
lently linked to a polyglycerolphosphate backbone which may carry carbo- 
hydrate and D-alanine subst 1 Clients (2). Teichoic acids (TA) , however, 
are never associated with cell membranes; they lack the terminal glyco- 
lipid coupling, and they may have a backbone of either pol ve Ivcero 1- 
phosphate or polyribitol phosphate (2). LTA may be converted function- 
ally to polyglycero.l TA by spontaneous deacylation in an aqueous environ- 

1 
merit, or mild alkaline, or acidic hydrolysis (107). The molecular 

weight of LTA (93) is probably between 3000-12000 but because of its 
tendency to form micelles in an aqueous environment, the apparent: mole- 
cular weight as determined by gel filtration is approximately four rail- 

2 
lion (110). Because LTA possess the glycolipid moiety, they are amphi- 

pathic. molecules exhibiting a propensity to spontaneously associate with 

proteins and biological membranes (103). Mammalian red bleed cells can be 

"coated" by spontaneous adsorbtion with an LTA containing extract and the 

cells can subsequently be aggluC i natcd with an anti-LTA scrum. Passive 

hemagglutination (PHA) performed in this manner with sheep reel b 1 ood 

cells has previously been reported by inanv invest ! gators who discovered 



Personal cormmin lea L ions from U. Craig, I'ept . of MC'S, t'niv. oi >'!.; k. 
Knox and A. J. Wieken, Institute for Dental Research, Sydney, Australia; 
and personal unpublished data. 

Data supported bv personal experience (see Figures M and 12). and 
personal communication from R. Craig. 



1.3 



erythrocyte-sensitizing antigens in coll free saline washings or spent- 
culture fluid from several gran positive organism? (101.102). Thesp so 
called "Rantz antigens" were recently shown to possess properties asso- 
ciated with LTA (111). Because only acylated LTA will hind to erythro- 
cytes, PHA provides a means of quantitating the amount of LTA in a 
preparation without having to contend with deaeylated TA contamination. 
The biological role of TA and LTA to the microorganism has been a 
subject of considerable disputation by several invest i gators in recent 
years. Thus far, at least three roles have been tentatively assigned: 
1). TA and LTA seem to function as "carrier" molecules for membrane 
and cell wall components, i.e. arrtphipathic LTA may be used by the cell 
to transport needed hydrophobic molecules through hydrophilic zones 
which would otherwise pose an almost impenetrable barrier. Fielder and 
Glaser have established that intracellular LTA serves as a lipid carrier 
for the biosynthesis of cell wall ribitol Leichoio acid in Staphylococcus 
aureus (112,113). Chaterjee and Wong (114) have demons t rated that LTA 
may serve as the acceptor in which nascent pept idoglycnn polymers are 
synthesized. 2). LTA seems to be involved in cell wall division and 
regulation. lloltje and Tomasz. have reported that LTA exhibits an inhi- 
bitory effect o\\ the function of autolytic enzymes during the division 
cycle of pneumococcus (115). It is interesting to note that similar 
functions have been described by Cleveland, et al. working with a strain 
°f Streptococcus f aeca l is (116.117,118). In these systems, LTA is 
deaeylated and released into the environment as TA. Once the concentra- 
tion of LTA is sufficiently levered, or the concentration of autolytic 
enzymes is sufficiently elevated, cell wall autolysis begins at the divi- 
sion rone. This autolytic activity then allows for Insertion of additional 



\h 



cell wall material. 3). LTA or TA may contribute Co the overall elec- 
trostatic charge of gram positive organisms. Although membrane 
localized, the long polar tails of many LTA penetrate the thick pepti- 
dogiycan layer and become externalized (107). These, together with the 
TA which are covalently Jinked to the cell wall (108) present a myriad 
of antigenic faces to the external environment C !.l c >, 120) . This antigenic 
presentation is of serological import since these antigens are often 
genus, species, group, or type specific (103,120). In addition, these 
polar tails generate a net negative charge by exposing the phosphate 
groups of the polyglyceroi or polyribitol backbone. This net negative 
charge has been teleological.lv assigned the function of maintaining elec- 
trostatic repulsion and dispersion of the bacterial cell (121). Since 
LTA has been shown to sequester certain cations such as magnesium (122), 
an additional function as a site of divalent eat ionic, convergence has 
also been postulated. The association with magnesium ions appears to 
be more than casual since protoplasts of Lac tobacil lus casei placed in 
a magnesium ion free or chelated medium rapidly lose their 1 , 1'A from the 
cell membrane. 

Anti-LTA titers (of both the IgM -and \rG classes) have been regularly 
reported in mice, rabbits, and man (2,125). Several clinical studies 
have reported Increases in anti-LTA titer- — including antibodies of the 
class LgA— rafter acute gram positive infections (12-'t,'2S). Pigs* guinea 
pigs, and rats exhibit a low level oi natural immunity to 1,1'A and recently, 
there have been reports of salivary fgA production as a result of gastric 
intubation of monkeys with Streptococcus mut nils (>7I'S so.r<->type ('. There 
is no doubt that TA and I TA of all gram positive genera thus far inves- 
tigated contain .antigenic moieties and that under certain circumstances 



LTA can be immunogenic (2). (if particular interest is the fart that the 
attachment: of streptococcal LTA to erythrocytes could he revcrsihly 
transferred from the erythrocytes to other tissue cells (104,12ft). The 
possible significance of this " trans ferabi 1 Uv" in relation te rheumatic 
fever and glomerulonephritis and pigeon breeders disease has been pre- 
viously discussed (9,104-106). However, despite this precedence the 
significance of the binding of LTA to oral epithelial cells in gingival 
pockets has not yet been investigated. Not only docs LTA mediate bone 
resorbtion as previously indicated, but spontaneous hybrid micells of 
LPS and LTA are known to occur, thus compounding the possibility of in 
situ immunological modulation. There is little doubt of the availability 
of extracellular LTA in this environment— Streptoco ccus mutans BUT alone 
has been reported to produce excess of 50 ng of LTA/ml in culture media 
(20). Recently, Wicken and Knox have studied the excretion of extra- 
cellular LTA from this organism in a chemostat under steady state loga- 
rithmic growth conditions. Results indicated that a generation time of 
10-14 hours (estimated to reflect that actual in vivo growth rate of this 
organism in the oral cavity) produced the maximal amount of extracellular 
LTA (1). Considering its ubiquity and the cariogenic nature of Sj^repjto- 
coccus mutans BUT (127- 1.30), the secretion of copious amounts of biologi- 
cally active LTA into the oral cavity has the potential of considerable 
influence en the host-parasite relationship. 

The objectives of the project were then defined as follows: 
(I) To establish if an LTA-cotU a in i ng extracellular extract of 



Personal communication of A. .! . Wlckett. 



16 

Streptococc us muta ns BUT was capable of inhibiting complement mediated 
cytolysis of target sheep erythrocytes . 

(2) To purify the extracellular LTA of S. mutans BUT to homogenlty. 

(3) To describe the nature of any ant i-comp 1 ementnry activity 
that purified extracellular LTA may exhibit. 

(4) To determine the site of action of any such inhibition. 

(5) To determine the mechanism by which purified extracellular LTA 
may enhibit anti-complementary activity. 

(6) To determine if the LTA from other gram positive genera and 
species can be shown to demonstrate anti-complementary activity. 



1ATER1ALS AND METHODS 



2 



Crude extracellular LTA ( LTAc x) ■ The initial studies were 
carried out utilizing LTAcx prepared in Australia by the method 
of Wicken and Knox (110). St repto coccus mutans BUT was grown 
to late stationary phase in a New Brunswick Microfirm fermentor at 
37°C. under anerobic conditions (95° N and 57, CO,,) in a complex 
medium. 

hater experiments utilized LTAcx prepared at (Gainesville, 
Florida. The original method was modified as follows. A Pell icon 
Cassette system (Millipore Corp., Bedford, MA) equipped with S.f) ft 
of PTGC filter material was used to dialvxe Todd-Howitt broth (Difc.o 
Laboratories, Detroit, Mi). A 100 ml culture of early log phase 
—' m ut:an s BHT was inoculated into 10 liters of dialyzod medium 
and incubated at 37° for 24 hours. The cells were harvested using 
a Delavai Cyrotester (Poughkoeps ie , NY). The supornate was passed 
through the Pellicon Cassette system (loaded with 1.0 ft of 0.45 u 
microporous membrane) to remove remaining cells and debris. The cell- 
free spent fluid wns then fractionated and concentrated by passage 
through 5.0 ft" PTGC membrane (nominal molecular wight exclusion 
limit of 10,000). The 1 [Iter retentato was washed in s Uu with several 
liters of water, collected and 1 voph i I i zed . The f reexo-dr ied retentato, 
designated as LTAcx. was stored in a tfess i en t or at -20°C. 

Solutions for complement assays. Isotonic Veronal buffered 
sodium chloride (VRS) , dextrose gelatin Veronal buffer with added 



CaCl, ; and MgGl- (DGVB), KDTA containing Veronal buffer (0.04 M 
EDTA-GVR) and gelatin Veronal buffer with added CaCl n and MgCl 
(GVB) were prepared as previously described by Hoffmann (131). 

Human com plement (HuC) . Fresh human blood samples were obtained 

from the Gainesville Plasma Corp., Gainesville, FL. The blood was 
allowed to clot at room temperature for about 60 minutes, and 
the serum was separated by cen trifugation at 500 X g at U C. The 
serum was collected and stored at -70°C. 

Guinea pig comp lement (GPC) . Fresh frozen guinea pig complement 
was purchased from Pel Freeze Laboratories (Rogers, AR) . The .serum 
was shipped in dry ice and it was stored at -70'"C after arrival 
in the laboratory. 

Complement components. Purified guinea pig CI and C2 were 
prepared according to Nelson et al. (132) and Ruddv and Austin 
(133,134). Functionally purified guinea pig C3, Cfl and C9 and 
human CI, C5, C6 and C7 were purchased from Cordis Laboratories 
(Miami, FL) . 

Erythrocytes ( E) . Sheep blood was taken by venipuncture from 
a single animal maintained at the animal research laboratory of the 
.J. Hillis Miller Health Center (Gainesville, FL) . One hundred 
milliliter volumes of blood were collected in equal volumes of 
sterile Alsevor's solution (135) and the blood was stored at 4"C 
for up to three weeks. 

Antibody sensitized slieeji ?£yj:jm\cvj:es (EAJ . Rabbit anti- 

sheep E stromata was obtained from Cordis Laboratories (Miami, FL). 
Sensitization of washed sheep F. was performed as recommended by 
the supplier. 



Complem ent component interme diate j^qnipj^exes. She op E in 
various stages of complement fixation were used in this study, 
EACI, EAC14 and EAC.U2 were prepared by methods described by Borsos 
and Rapp (136)'. EAC1423567 were prepared by the procedure described 
by Hoffmann (137). Unless otherwise indicated, guinea pig CI, 
C8 and C9 were used in all instances, and the remaining C components 
were from human serum. 

Treatment of cells and _cenular^^termccyji^cti_ with LTAcx . 
Unless otherwise Indicated, cells were washed and suspended in VBS 
at a concentration of 10 9 /ml. Equal volumes of these cells and 
LTAcx were mixed and incubated at 37' J for 20 minutes with continuous 
shaking. The mixture was then placed in an ice bath for 10 min- 
utes. At the end of incubation DCVB was added to the mixture and 
it was centrifuged at 500 g for five minutes. The suoernate was 
discarded and the cells were suspended and washed thrice with 
DGVB (0° for 10 minutes at 500 g) to remove any unbound material. 
The cells were then resuspended in DGVB at a concentration of 10 /ml. 
A sample of the cells were tested for cell-bound LTA using passive 
hemagglutination with rabbit anti-LTA. The remaining cells were 
used in experiments to detect acquired resistance to hemolysis. 

Passiyo Jiema^lutJ_na>_iqiLl0iA2- P;lss [ ve hcma ^ 1 nt Lnat L ° n Wa " 

carried out using a micro., i t rat ion system. Fifty uL of a VBS 
dilution of anti-LTA were added to the first row of wells of a round 
bottom microtiter plate (Cook Engineering Co., Alexandria, VA) 
and 25 ui (one droo 'rem the calibrated pipetes supplied with the 
system) of VRS were added to the other wells on the plate. The 
anti-serum was serial lv diluted In situ and one drop of LTAcx 



20 



treated cells was added to each well. Controls for spontaneous 
or nonspecific agglutination consisted of wells that contained anti- 
serum and sheep E which had never been exposed to LTAc.x. Treated 
sheep E plus VBS constituted another control. The microtiter plate 
was incubated at 37°C on a Cordis Micromixer (Cordis Laboratories, 
Miami, FL) for 15 minutes. The plates were removed from the mixer 
and the cells were allowed to settle for two hours at 37°C, followed 
by three hours at room temperature. 

Modified passive hemag glutination (PHA g) ■ A modification of 
the above technique was used to semi-quant itate the amounts of LTA 
present in various preparations. The same apparati were used, but 
instead of antibody, LTA-containing extracts were added to the 
bottom wells and serially diluted in situ as described. After 

Q 

each LTA source was diluted, one drop of sheep erythrocytes (10 /ml 
in VBS) was added to each well and the plate was then incubated 
at 37°C for 20 minutes and at 0°C for 10 minutes. The ceils were 
kept in suspension by vibrating the plate on a Cordis Micromixer 
during both incubation periods. One drop of GVB was then added to 
each well and the plate was centrifuged at 200 g for 5 minutes. 
The entire plate was then abruptly inverted over ahsorbant paper 
towels and allowed to drain for approximately one minute. One 
drop of GVB was again added to each well and the plate was vibrated 
at 0°C for S minutes to resuspend the pellet. An additional drop 
of GVB was added per well and the plate was again centrifuged at 
200 g for 5 minutes. This washing procedure was repeated throe 
times and the cells were then finally resuspended in one drop 
of GVB. One drop of antl-LTA (diluted 1:1000 in VBS) was then 



added to each well and the plate was incubated at: 37°C for 15 

minutes on a Cordis Micromixer. The plate was removed from the 

mixer and the cells were allowed to settle for two hours at 37°C, 

followed by three hours at room temperature. 

Inhibition of complem ent, med ia ted l ysis. EA coated with 

LTA (EA ) were tested by mixing 0.1 ml of EA (10' rells/ml) 
L I A I j 1 A 

in DGVB and 0.4 ml of DGVB diluted HuC. The HuC was diluted so 
that a maximum of 80 percent lysis was produced in EA which had 
not been treated with LTAcx. The mixture was incubated at 37°C 
with continuous shaking for 60 minutes. One milliliter of ice 
cold EDTA-GVB was added, the mixture was centrifuged for 5 minutes 
at 500 g at 0°C and the supcmatent fluid was recovered. The 
optical density of the supernatent fluid was determined at a wave 
length of 414 nm. Inhibition of hemolysis was calculated for each 
concentration of LTAcx used by comparing the extent of lysis in 
each assay with a control reaction mixture which contained LA 
that had not been treated with LTAcx. 

E ffe c t of LTA cx on the titer of antibo dies specific L _Cor _g_heei? 
eryth rocyte stromata . Because LTA associate with some proteins (138) 
it was necessary to perform a hemolytic antibody titration to 
determine if the abilitv of the immunoglobul Ins to fix complement 
at the cell surface was being affected by LTAcx treatment. The 
possibility of similar antigens in LTAcx and sheep erythrocyte 
stromata was also considered. bquil volumes of LTAcx (500 ug/m! 
in VBS) and rabbit anti-sheep E stromata were incubated together 
at 37°0 for 20 minutes. A control consisted of incubating an 



01 



equal volume mixture of VRS and' anti-sheep erythrocyte stromata 
for the. same time at the same temperature. The antibodies were 
then titrated using limiting amounts of complement (135). 

C I fixation and tran sfer. The number of CI molecules hound 
to an antigen-antibody complex can be measured by the CI fixation 
and transfer test described by Borsos and Rapp (139). In a mod- 
ification of this procedure, an attempt was made to quantitate 
the number of CI molecules fixed to EA which had previously been 
treated with LTApcx. Buffer controls and EA were prepared 
as previously described, and after washing were resuspended at 

Q 

10 cells/ml in DGVB. Equal volumes of EA, „. and EA were in- 

LTA VBS 

cubnted with CI at 30 D C for 15 minutes. The cell mixtures were 
washed twice with DGVn, and resuspended in CVB at a cell concen- 
tration of 1 X 10 /ml, 5 X 10 7 /ml, and 1 X If) /ml. One volume 
of each cell concentration was added to one volume of EAC4 (at 
i X 10 cells/ml) to permit transfer of CI from EA CI to EAC4. 
The cells were incubated at 10°C for 15 minutes, and then C2 and 
C-EDTA were added in relative excess as described previously. 

A variation of the CI transfer assay was performed by treating 

preformed EAC1 with LTA or buffer control as described. The 

8 
resulting EAC1 were resuspended to 1 X 10 cells/ml in GVB and 
x ' 

the amount of CI capable of transfer was measured as described 
above. 

Go 1 f ij. t_rat_ion . LTAex was fractionated on a ?. . 5 cm X 100.0 cm 

column of Bio-Gel A-'jM, 200-400 mesh (Biorad Laboratories, Richmond, 



Cn this instance, "x" represents LTA or the appropriate buffer 
treated control. 



CA) using a modification of the method described by Wtcken and 
Knox (110). The column was equilibrated and eluted using 0.01 M 
Tris carbonate (Sigma Chemical Co., St. Louis* MO), pil 6.8. 

Hydr opho bic Affini t y Column chromatog raphy. Because of the 
hydrophobic nature of the fatty acid moieties of lipotelchoic acid, 
adsorbtion to a stationary phase of a chromatographic column was 
used in an attempt to further purify the LTA. LTAppK in buffer A 
(0.01 M Tris carbonate pH 6.8, 1,0 M Nad was loaded on a 25.0 X 2.25 cm 
column packed with Octyl Sepharose (Pharmacia Fine Chemicals, 
Piscataway, NJ) and equilibrated in the same buffer. After e Luting 
with 150 ml of starting buffer A, the reservoir was then changed 
to buffer B (0.01 M Tris carbonate pH 6.8) and another 100 ml were 
eluted. Buffer C consisted of 250 ml of a gradient ranging from 
10-70 % propanol (by volume) in 0.01 M Tris carbonate, pH 6.8. 

Octyl Sepharose is a derivative of the cross linked agarose 
Sepharose CL-4B. The terminal n-octyl groups of this agarose gel 
confer a hydrophobic i ty to the matrix. By exploiting this property 
it was hoped that polar or neutral non-interacting components 
would be removed by elution with solutions of high ionic strength. 
The lipotelchoic acid would then be eluted from the matrix with 
an organic solvent such as propanol. (It is imperative that all 
tubing, connections and gaskets used throughout the column be 
constructed of a material that is resistant to organic solvents). 



This method represents a modification of a procedure described 

by A..!. Wicken and K. Knox (Sydney, Australia) via personal commun- 

icat ion. 



Removal o f salt a nd propan ol from LTA containing e xtra cts. 
Removal of salts and/or propanol from various preparations was rapidly 
and quantitatively accomplished by pel filtration utilizing LII20 
(Pharmacia Fine Chemicals, Piscatawav, NJ) as the solid phase support 
matrix. The most commonly employed column was 50.0 cm X 2.5 cm hut 
a larger 65.0 cm X 3.0 cm column was sometimes utilized. The column 
was packed and equilibrated with deionized water. Sample preparations 
usually involved rotary flash-evaporation (Buchlet Instruments, Fort 
Lee, NJ) in order to reduce the volume of sample to 15-20 ml. Elution 
of product was carried out at a pressure head of approximately 50 cm 
water and approximately 4.0 ml effluent were collected per tube. 

Phosphatid yl choline vesi cle (PCV) purification of _LTA — 
(a) Pre paration of PCV. Although renorted as the method of choice by 
other investigators, in our hands Oetyl Sepharose purification 
of LTA from Streptococcus mutan s BUT resulted in a product still 
highly contaminated with polysaccharides. Tn an attempt to achieve 
homogeneous purification of LTA, a modification of the above mentioned 
hydrophobic adsorbtion principle was employed. In this procedure, 
artificial membrane vesicles were prepared with 1)1, mhospha tidy 1 
choline dipalmitoyl (PC) (Sigma Chemical Co.) as the sole constituent 
via a modified method of Hill (U0) . In brief, iO.O mg of VC was 
placed in each of several SO ml high speed glass Corex centrifuge 
tubes (Corning Glass Works, Corning, NY) and dissolved with one ml 
chloroform, The solvent was gently evaporated in a 50°C water bath 
while rotating the tubes so as to coat the bottom 5 or 6 cm of the 
tube with PC. Once dry, the tubes were placed in a lyophilUnt ion 
flask and any residual solvent was removed in vacuo. One milliliter 
Wichen, A.J.-, and Knox, K. — Personal communication. 



2 5 



of 0.01 M Tris carbonate pll 6.8 was then ridded to each tube and they 
were placed in a 50°C water bath. Once warmed, the tubes were 
vigorously vortexed (Vortex Genie Mixer, Scientific Industries Inc., 
Bohemia, NY) and the cycle of warming and vortexing was continued 
until a milky emulsion was formed. Fifteen milliliters of 0.01 M 
Tris carbonate were then added to each tube and the tubes were centri- 
fuged at 27,000 g for 30 minutes. The supcrnatent fluids were then 
decanted, the pellets were resuspended in 1.0 ml Tris carbonate 
buffer and warmed to 50°C in a water bath. The tubes were gently 
swirled (but not aggitated) to dissolve and resuspend the pellet . 
The resulting phosphatidyl choline vesicles (PCV), devoid of very small 
vesicles, were then used to adsorb LTA from LTAppx. 
(b) Preparation of FCV-LTA. Two milliliters of LTAppx at a concen- 
tration of 1.5 mg/ml in 0.01 M Tris carbonate, pH 6.8 were added to 
each centrifuge tube containing 1.0 ml of PCV. The tubes were covered 
with parafilm (American Can Co., Neehaw, WS) and incubated for 
90 minutes in a 37° shaker water bath. Thirteen milliliters of 
0.01 M Tris carbonate were then added to each test tube and they were 
centrifuged at 27,000 g for 45 minutes. The supernates were discarded 
and the pellets were gently resuspended in 1.0 ml of Tris carbonate 
buffer at 50°C as previously described. 

Fifteen milliliters of buffet were then added to each pellet, 

the tubes were gently swirled and then centrifuged as described. 

The pellets were washed three times in this manner. The final pellet 

was drained and then dissolved in 5.0 ml of eh lorof orm/me tlianol 

(3 + 1 v/v). The tubes were then covered with aluminum foil and 

allowed to sit at room temperature for 60 minutes. 



?A 



A Millipore 15 ml analytical filter holder (Millipore Corn., 
Bedford, MA) was loaded with a 3.0 \\ fluoropore membrane (Millipore 
Corp.) and washed with several volumes of the chloro form/methane 1 
solvent. The test tubes were all sequentially decanted into the 
apparatus and the contents were allowed to filter bv gravity through 
the membrane. Each test tube was washed with several volumes of 
warmed chloroform and decanted into the filtering apparatus. Finally, 
the barrel and filter were washed in situ with warm chloroform. The 
filter was removed after air drying in situ and placed in LO.O ml 
of deionized water warmed to approximately 40°C. All centrifuge tubes 
and the barrel of the filtering apparatus were washed with warm 
deionized water and all products . were combined. The resulting 
product was passed through a 25 mm Swinnex filter (Millipore Corp.) 
loaded with a 5 \i microporous membrane (Millipore Corp.) to remove 
particulate debris. The membrane was washed i_n Situ with several 
volumes of warm deionized water. The filtrate was collected directly 
into a lyophi lization flask and was then shell frozen and lyopbilize.d. 

The final product was stored in a dessicator at -20°C. 

14 

C Phosphatidyl choline analysis . In order to detect any 

phospholipid contamination of the LTA throughout the previously 
described PCV purification, radioactive PC was used to label the phos- 
pholipids in the vesicles. Approximately 2.3 i;Ci (5 X 10 ' DPM) 

14 
of C labeled phosphatidyl choline (Amersham Searle Corp. , Arlington 

Heights, TL) were added to 40 nig of phosphatidyl choline dipalmitovl 

in a 30 ml. Corex centrifuge tube. Phosphatidyl choline vesicles 

were prepared from this and the non- labeled contents of three 

other tubes by I he method;-, previously described. Fifty microliter 



27 



14 
samples from the C contain ing test tube were taken at each st*»p 

of the purification and placed In empty glass sr. in filiation vials. 
The samples were heated to 50°C in a drying oven to remove the 
solvent from the sample. Once dry, 50 ul of chloroform were used to 
redissolve all samples and then 5.0 ml scintillation fluid containing 
toluene (scintillation grade, Mai linckrod t , St. Louis, MO), 0. V PPO 
(2,5 diphenyloxazole), and 0.01% P0P0P (1,4-di (2-(5-phenylo:<azoiyl)- 
benzene.) were added to each vial. The degree of C-PCV contam- 
ination of the final product was determined by placing the entire 
LTA-containing-fluoropore filter in a scintillation vial with 5.0 ml 
scintillation fluid. The possible influence of quenching by the 
fluoropore filter was investigated by adding equal al iquots of 

^C-PC to two scintillation vials one of which contained a fluoropore 
filter in addition to scintillation fluid. No appreciable difference 
in CPM was observed. Disintegrations per minute (DPM) values were 
calculated from a standard quench curve constructed for use with chloro- 
form. Standard ratios were determined for each sample and percent 
efficiencies were extrapolated from the standard quench curve. 1'his 
volume was then used to correct counts per minute (CPM) to PPM. Unless 
otherwise indicated, the samples were counted for 10 minutes in a Beckmnn 
LS-.133 liquid scintillation counter (Bookman Instruments, Fullerton, 0A) . 

Colormetric assays. Phosphorous was determined by the method 
of Lowry et al. (I'd) with absorbaneies measured at 820 nm. iota! 
carbohydrate was measured by the phenol sulfuric acid assay as des- 
cribed by Dubois et al. (142). Total protein was performed on samples 
using the Rio-Kad Protein Assav (Bio-Rad Laboratories, Rockvitle Center, 
NY). Samples and the standard curve were prepared following the 
manufacturer's recommendnt ions. 



28 



GgJLi , i g u i d C h r orc a t o g r a p h y . Carbohydrate analysis was performed 
after treatment of the samples with 1.0 N H,SO, in sealed ampules 
for 8 hours at 105°C. Upon cooling, the seal was broken and exactly 
0.2 ml of mannitol (either at: 5.0 mg/ml or 1.0 mg/ml depending on 
carbohydrate concentration of the sample) was added as an internal 
standard. The contents of each vial were quantitatively transferred 
to 15 ml centrifuge tubes (Corning Glass Works) containing 0.3 g 
BaCCy Each centrifuge tube was heated in a boiling; water bath 
and alternately vortexed until the pH approached neutrality as 
indicated by full-range pH paper (Micro Essential Laboratory, 
Brooklyn, NY). All tubes were centriufgcd at 500 g for 5 minutes 
and the supernates were removed and collected in appropriately labeled 
13 mm screw cap tubes fitted with teflon lined lids. The centrifuge 
tubes containing BaCO were washed once with one ml of deionic.ed 
water and the supernates were appropriate! v pooled. 

After Ivophiliza tion , the hydrolyzed carbohydrates were con- 
verted to trimethylsilyi ester (TMS) derivatives by the addition 
of 0.2 or 1.0 ml (depending upon carbohydrate concentration) of 
TRI SIL Z (Pierce Chemical Co.). Samples wore warmed to approx- 
imately 60°C in a water bath for 15- JO minutes before use and 
assayed using a Packard 800 series gas chrnmatogrnph equipped with a 
flame ionization detector. The gas chromatographic column (153 cm X 
4 cm) wis packed with RE-40 (1LTRAPHASK 3.". on Chromosorb W (HP) 80/1.00 
mesh matrix (Pierce Chemical Co.. Rockford, II, ). Column and detector 
temperatures were set at I60"C and l c )5T respectively. The N„ carrier 
gas was set at approximately 30 ce /minute. 



29 



Amino acid an alyses. Amino acids and amino Bugnrs were measured 
on a JEOL model JLC-6AH automated amino acid analyser (JEOL, Inc.. 
Cranford, NJ ) . Sample hydrolysates were prepared as described by 
Grabar and Burtin (143). 

Cll». c ^ and Cls puri fication . Highly purified human Clq 
was prepared from whole human sera by the method of Yonemasu and Stroud 
(144). Highly purified human Cls and Cls were prepared by a minor modi- 
fication of the method described by Saka i and Stroud (35). For the final 
resolution step, Bio-Rad Cellex-D DEAE with binding capacity of 1.07 meq/g 
(Cellex-D, Bio-Rad Laboratories, Rockville Center, NY) was substituted for 
fibrous DEAE cellulose Whatman DE-23. The DEAE was washed and prepared 
according to the manufacturer's specifications. Final elution of the pro- 
duct was accomplished with (he use of the same, eluting buffer is described, 
but instead of a stepwise elution of the column, an ionic gradient from 
0.2 - 0.4 RSC (relative sodium chloride concentration) was utilized. 

Disc acrylamide gel e lectrophoresis of Clci^, Cls and Cls. This was 
carried out essentially as described by Yonemasu and Stroud (144) but with- 
out the use of sodium dodecyl sulfate (SDS). 

Cls inhibition as says. The ability of Cls to consume C2 activitv was 
assayed by a modification of the method described by Sakai and Stroud (35). 
Briefly, 0.1 ml of Cls (approximately 8.0 X 10 site forniiny; units, STU/ml) 
plus 0.J ml LTApcx (100 (l g/m.l in DVB) were Incubated at 30" for 1.5 minutes. 

One tenth milliliter of C2 was then added at a concentration of approxi- 

7 
mately 9.0 X 10 effective mo 1 ecu 1 es/ml and incubated at 37T for 30 min- 

utes. At the end of the incubation, 9.7 m! cold DOVB were added to the 

mixture resulting in a 1:1.00 dilution of the C2. The C2 was then serially 

diluted and 0.1 ml aliquots from each dilution were added to 0. I ml of 



30 



EAC14 (10 cells/ml in DGVB) . The mixture was intubated at 30'-' C for 10 
minutes and cooled to 0°C in an ice bath for 2.0 minutes. Three tenths 
of a milliliter of C-EDTA (1:37.5 in 0.04 M EDTA-GVB") were then added to 
each test tube and the mixtures were incubated at 37°C for £0 minutes. 
At the end of the incubation period, 1.0 ml of cold EDTA-GVB was added, 
the. tubes were centrifuged, and the supernates read for release of oxy- 
hemaglobin at a wave length of 414 nm. External controls consisted of C2 
with no Cls nor LTApcx, C2 with Cls but not LTApcx, and C2 with LTApcx 
but no Cls. The usual internal controls (spontaneous lysis, color cor- 
rection, no C2, and total lysis) were included at all times. Results were 
expressed as percent inhibition of C2 consuming ability compared with a 
control containing only Cls and C2. 

The ability of Cls to hydrolize the synthetic substrate p-Tosyl-l- 
arginine methylester (TAMe) was determined as described by Magaki and 
Stroud (38). Inhibition assays were performed by incubating equal volumes 
of Cls (approximately S.O X 1.0 SFU/ml) and LTApcx (approximately L00 uR/ml) 
at 37° for 10 minutes. Residual Cls activity was then determined as des- 
cribed (38-40). 

Clq inhi bition as says. The effect of LTApcx on the ability of puri- 
fied Clq to bind to antibody sensitized sheep erythrocytes was determined 
by methods described by Loos et al. (34) and Raepplc ct* al . (40). Fqna ! 
volumes of Clq (approximately 1.3 X 10 SFU/ml) and LTApcx (10 ug/ml ) 
were incubated at 37° for 10 minutes. Residual Clq activity was then 
determined as indicated above. 



RESULTS 

Inhibition of wh ole human co mplement ...j^jL- c -^J^-.i ? - : ?- L - r -^£.pJJj:L^i r . 
lipoteich oic acid (LTAcx) . To determine whether LTAcx had any effect on 
whole human complement, equal volumes of LTAcx and whole human complement 
were preincubated at 37°/30 minutes. After pre incuabt ion, the complement 
source was serially diluted in DGVB and the residual hemolytic activity 
was titrated. As shown in Figure 1, approximately 502 of the whole com- 
plement hemolytic activity (measured in CH-- units) was consumed. Further- 
more, as seen in Figure 2, this consumption was dependent on the concen- 
tration of the LTAcx used. 

T i t r a t ion o f complemen t components ju_j^^l^^hujn^n_gj_ra__at"ter treat- 
ment with LTAc x. One mechanism for fluid phase consumption of whole com- 
plement could have been the interaction of natural antibodies in the human 
sera with LTA or some other antigenic substance in the crude extract. The 
result would be the fixation of CI and subsequent activation of Ca and C2 
via classical pathway. Another explanation for decreased hemolytic activ- 
ity could have been the activation of the alternative pathway in a manner 
analogous to LPS. To differentiate between these two modes o\ activation, 
individual component titrations wore performed on human sera incubated 
with LTAcx. Tn addition, 03 titrations were carried out in the presence 
of ethyleneglycol-bis (l< Amino Ethyl Ether) N,N tetraacetic acid (F.OTA) 
and Mg ions. This chelating agent preferentially Hinds Ca ions (US.HM, 
and by reinforcing the FGTA buffer with Mr ions one can effectively deplete 
the available Ca ions yet maintain relatively high levels of Mg ions. 
Thus, the Ca ion dependent classical pathway is blocked, but the 
alternative pathway can function relatively unimpaired ( L'> "> , 1 W ) . 



Figure 1. Titration of whole human complement after incubn t Ion 
with crude extracellular lipoteichoic acid (LTAcx). 
Symbols: (o) Non-treated control: (•) Serum treated 
with LTAcx at 500 pg/ral . 



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Figure 2. 



Dose response Inhibition of whole human romp lenient 
after incubation with varying concent rn t ions of 
LTAcx. The non-treated control is abbreviated 
as NTC. 



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A typical component titration in scrum treated with LTAcx is 
depicted in Figure 3. In this example, lite LTAcx treated serum was 
serially diluted in DGVB- Next EAC142, C5, f>, /, and C8-9 were added 
sequentially to the dilutions. Since all components were added in 
excess, C3 became the limiting factor in contributing to the hemolysis 
of the target cells. Percent lysis in each test lube was mathematically 
converted to Z (the average number of SAC 14 23 sites per cell) and this 
was plotted against the reciprocal of the serum dilution. Percent inhi- 
bition of site forming units (SFU) was then calculated from Z=] values 

or percent inhibition of CH-„ units was determined from values asso- 

50 

ciated with Z~ Q.&9. Figure 4 represents a composite of multiple com- 
ponent titrations from whole human sera treated with I.TAcx. As can lie 
seen in this figure, CI and C4 activities were consumed to some degree, 
however, more than 50% Inhibition of C2 activity was observed. As 
indicated, C3 activity was also consumed during preincubation of com- 
plement with LTAcs, but incubation with purified (") ' produced no inhi- 
bition of C3 hemolytic potential. No C3 consumption occurred if the 
incubation was performed in the presence of the (die later ethy lenediamine 
tetra acetic acid (RDTA) and Less than 7'"' if incubated in the presence 
of Ef.TA-Mg ions. The above results indicated the necessity for divalent 
cations as eofactors mediating the consumption of C3 in the presence of 
LTAcx. In addition, there appeared to be a requirement for other serum 
factors (possibly natural AB and/or components of the alternative path- 
way) since purified ('.') activity remained unaffected l>v tncuhnfion with 
LTAcx. 



Figure 3. 



Titration n£ Cl in whole human serum after treatment 
with LTAcx. Symbols: (*) Non-treated control; ((O 
Serum treated with LTAcx at a concentration of 2 r >fl 

Mg/ml. After incubation, sera were titrated for 
residual C3 activity according to procedures 

described in Materials and Mo (hods. 



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Figure 4. Complement component titration of whole human sera 

after treatment with LTAcx. The sera were incubated 
with the LTAcx (500 ug/ml) then titrated for resi- 
dual activity of the components indicated as described 
in Materials and Methods. 



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Inhi bition of ^»^p lemgn^_lv_sis_of_ LTAcx trea te d EA . During an 

experiment In which EA treated with LTAcx were tested for reactive 
lysis, it was discovered that the LTAcx treated cells exhibited Less 
hemolysis than even the buffer treated controls. This serendipitous 
observation led to the discovery that LTA treated EA were refractory 
to complement mediated lysis. To confirm these results, various concen- 
trations of LTAcx were used to treat EA. After the treated cells were 
extensively washed they were tested for their susceptibility to lysis 
by complement. The same cells were also tested for the presence of 
cell-bound LTA using the passive hemagglutination technique (PHA) with 
anti-LTA. The results shown in Figures 5 and 6 indicated that both 
the extent of inhibition of hemolysis and PHA titers were LTAcx dose 
dependent . There was a decline in both activities only after the LTAcx 
had been diluted to a concentration of 62.5 ug/ml . The decrease in 
titer below this concentration indicated that the test cells were no 
longer saturated with LTA. There was a concomitant drop in inhibition 
of lysis at 62.5 ug/ml. EA which were treated with uninoculatod cul- 
ture medium (dialyzed Todd-Uewitt broth) were unaffected when oomple- 
emnt was added. 

Effect of LTAcx on lysis of s heen E and sheep E eeJJLuJ.ar 
i nterme diates. The treatment of LA with LTAcx caused the cells to 
become relatively resistant to complement mediated lysis. This could 
have been due to an effect on the antibody molecule.':, an effect on one 
or more of the complement components, c^r an alteration of the cell 
membrane . 

To further investigate the nature of the complement Inhibition 
associated with LTAcx, sheep K, sheep EA , and various sheep E complement 



Figure 5. Inhibition of complement mediated lysis of EA 
treated with varying concentrations of LTAcx. 




SISA1 dO NOiilQlHNI !N3Dd3d 



Figure 6. Passive hemagglutination (PIIA) of EA ttrcnted with 
varying concentrations nf I/L'Ar.x. 





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component intermediates wore treated with LTAcx and analyzed for sus- 
ceptibility to complement mediated lysis. The LTAcx treated cells were 
also tested for bound LTA using PDA with anti-LTA. Results indicated 
that !i, EA, and EACH were all refractory to complement mediated lysis 
and that LTA was detectable on the surfaces of the cells (Figures / and 
3). However, EAC 142 3567 which had been treated with LTAcx were not re- 
sistant to lysis despite the fact that LTA was detectable on the cells 
(Figure 8). Thus, the inhibitor appeared to affect a complement com- 
ponent required for lysis os EAClT, but which was unnecessary for Lysis 



of EAC1423567. 

In an attempt of focus on the site of inhibition, the ability of 
LTAcx to affect the hemolytic susceptibility of EAfU42 was examined. 
This intermediate possesses C3 convertase activity (C42) which is in- 
volved in the generation of SAC1423 and SACU215. However, CI is not 
required for lysis of the intermediate once SA042 have been formed (148). 
Failure of LTAcx to inhibit this intermediate would indicate that CI 
convertase was not the step in the complement sequence affected by the 

LTAcx. 

Sheep EAC142 were treated with LTAcx according to the protocol 
that has been described. For this experiment, three different amounts 
of C2 were used to generate EAC142 from RACU. The results clearlv 
indicated that there was no inhibition of the intermediate complex 
EAC142 (Figure 9). testing by PHA with antibodies specific for LTA 
confirmed the presence ol LTA on the surfaces of the cells at the same 
relative concentrations found when the other intermediate complexes 

were tested. 

Effect of LTAcx on an 1 L-shec^J^throcj jtejmt_Uwdles. Some 



Figure 7 . 



Effect of LTAcx treatment on the lysis of various 
complement component intermediates. Each cellular 
intermid Late was prepared and then treated with 
LTAcx (125 yg/ml) . Lysis was developed using 
procedures described in Materials and Methods. 
Percent inhibition was calculated by comparison 
against buffer treated controls. 



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Figure 8. PHA of various LTAcx treated complement component 
intermediates . 



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substances in LTAcx might be capable of interacting with the anti- 
bodies used to sensitive sheep E. This interaction could then lead to 
an impairment of CI activation and result in reduced lysis. Such a 
mechanism might be the reason why E and MA become resistant t.:o lysis 
after treatment with LTAcx. Therefore, antibodies to sheep erythro- 
cyte stromata were incubated with LTAcx. The mixture was then diluted 
to the point where the LTAcx-related inhibition could not be detected 
and the antibodies in the mixture were titrated (135). It was found 
that antibodies that had. been preincubated with LTAcx had the same 
titer as antibodies that were incubated for the same time and temperature 
with VBS (Figure 10) . 

Part ial purification of LTA. Partial purification of LT.A and 
the complement inhibitor was accomplished by gel filtration of the LTAcx 
through an A-5M Biogel column. The results of a typical experiment are 
shown in Figure 11. Areas of antigenicity wore resolved bv immunodif- 
fusion in an agarose gel utilizing an anti-serum specific lor the LTA 
backbone. Fractions were pooled as indicated (A-F), and each pool was 
dialyzed against water and subsequently IvophUized. Note that pools B, 
C, and E contained high Levels of phosphorus and that the zones of anti- 
genicity were also located in these areas. Utilizing extracellular 
extracts from S. mutatis and other microorganisms, similar fractionation 
profiles under comparable renditions were obtained by Wiekcn and Knox 
(lit!) and lilewieis and Craig. Analysis bv these workers revealed that 
the second phosphorous containing peak (peak El) contained LTA whereas 
the trail in;; phosphorus peak contained deacvlated LTA and wall Leichoie 



Personal communication 



Figure 10. Effect of f.TArx on hemolytic antibody titration. 

Antibodies to sheep red blood cell strornata (Ah) wore 
incubated with LTAcx (SOO ug/ral) and residual hemolysin 
activity was titrated by procedures described in 
Materials and Methods. Lysis of cells was developed 
with whole guinea pip complement. Svmbols: (o) Ah 
incubated with I.TAex; (m) Ab incubated with buffer. 




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acids. Because peak II represented partially purified extracellular 
llpoteichoic acid, the recovered material was designated LTAppx. 

A sample of each pool was rehydrated to s ue/ml and reacted with 
EA, according to standard procedures (Materials and Methods). Each RA 
preparation was analyzed using the. complement inhibition assay and 
tested for bound LTA by PHA. Only the pools containing LTA (as demon- 
strated by PHA) caused inhibition of complement mediated lysis (Table 1). 

Despite the excellent separation of LTA from most of the materia] 
that absorbed light at a wave length of 260 nm, and presumably from 
all deacylated LTA or TA, two persistent problems arose with this puri- 
fication procedure: 

1). Polysaccharide contamination accounted for a major portion of 
the mass recovered in peak 11, and 

2). The total mass of LTAppx under peak II was almost immeasurah Iv 
small . 

In an attempt to at least increase the yield of peak II material, 
a Millipore Cassette system was employed to both concentrate and frac- 
tionate the spent culture supernate (Materials and Methods). This method 
of LTA enrichment proved highly successful as evidenced by the results 
in Figure 12. Even after values are corrected For the greater mass of 
crude extract applied on the latter column the mass yield of 1 I'Anpx was 
some fifteen fold greater than that obtained with previously employed 
procedures ( P igure | | ) . 

An analysts of results tracing the partial purification of I.TA 
is summarized in 'fables I and 5. It should be noted that the total 
amount of Pi, mass, protein, and A_, absorbing; material decreased 

2 n() 

several thousand fold In the purification process, whereas the total 



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Figure 11, Partial purification of LTA by A5-M gel filtration. 
Symbols: (•) A nbsorbance (max inn] absorbance 
wavelength for nucleic acids); (,-) A,-, absorbance 
(maximal absorbance wavelength for carbohydrates as 
determined by the Phenol Sulfuric Acid assav) ; (A) 
Pi concentration in n-moles as determined by the 
Lowry Pi assay; (+) Antigenicity as determined by 
Ouchter l.onv gel diffusion using .an antisera 
directed against LTA backbone. 




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Figure 12. Partial purification of LTA by A.5-M gel filtration 
with LTA enriched starting mterial. Svmbols: (•) 

A 260 absorha,l!: ' G: Co) Pi concentration in u-moiew/mi 
as determined by the Lowry Pi assay: (+) Antigenicity 
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Figure 9. Effect of LTAcx treatment on the lysis of EAC142. 
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were then treated with LTAcx (250 pp/ml) and lysis 
was developed using procedures described in Materials 
and Methods. Symbols: (o) KAHI.42 incubated with 
LTAcx; (•) BAC142 incubated with buffer. 





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amount of LTA in the sample, PHA titer, and percent lytic Inhibition of 
EA remained relatively unchanged or increased in value. 

Purifi cat ion of LTA by hydrophobic interaction gel chrom atography . 
A 25.0 X 2.25 cm column packed with Octyi Sepharnse and equilibrated in 
buffer A was prepared as described in Mate-rials and Methods. Approxi- 
mately 6.0 mg of LTAppx dissolved in 10.0 ml of buffer A were applied 
to the column. As can be seen in Figure 13, a small amount of phosphate 
containing material passed unimpeded through the column. A slightly 
greater mass of polysaccharide was also excluded without bind inc. No 
additional material eluted from the column with buffer B. Point C on 
the graph marks the location where a 10-70% propanol gradient was begun. 
Point D represents the point where a significant volume decrease per 
test tube was observed. Since fractions were collected on a "drops per 
tube" basis, the presence of propanol in the effluent causes a change in 
surface tension of the drop resulting in a decreased volume per drop. 
The ultimate result is a decrease in the volume per tube. This, test 
tube volume provided a convenient means ol monitoring the progress Of 
the propanol gradient. 

it should be noted that despite the use of a gradient (the original 
procedure called for a single step-wise elution with 50% propanol) 
significant amounts of carbohydrate elnted with the 1/1 A. As indicated 
on the the graph, all arms containing phosphates also contained LTA as 
detected using PHA. Tin- fact that a small amount of LTA passed unbound 
through the column suggests that cither t he column"* b hid inR capacity was 
exceeded, or perhaps the f.TA was only partially acviatcd and not capable 
of tenacious hydrophobic hind in;;. 



Figure 13. Purification of LTA by Octyl Sepharoso hydrophobic gel 
chromatography. Symbols: (•) A ahsorbance; (A) 
concentration of carbohydrate (n-molos/ml) as determined 
by the Phenol -Sul fur i c Acid assay. Concentrations were 
determined using glucose as a standard carbohydrate. 
(o) Concentration of Pi (n-rno 1 es/ml) as determined by 
the Lowry Pi assay; (+) zones of antigenicity as 
determined by PHA using antisera directed against LTA 
backbone; (A) elution with buffer A ( 1 . OM NaCl, 0.01 
M Tris-carbonate pH 6.8); (B) elution with buffer B 
(0.01 M Tris-carbonate, pH b.8); (C) elution with a 
10-70° gradient of a propanol-buf f er B mixture; (D) 
elution volume at which significant reductions of vol- 
ume/tube were observed, indicating elution of propane! . 



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All test tubes containing greater than 25.0 n-moles Pi/ml were 
pooled. The entire peak (approximately 22 ml) was loaded on to a 
65.0 cm X 3.0 cm column packed with LH-20 equilibrated with rieionized 
water. Four and two tenths milliliter of effluent were collected per 
test tube a a flow rate of approximately 30.0 ml/hour. The results 
of this procedure, which simultaneously removed salt and propanol, are 
shown in Figure 14. The column effluent was monitored at a wave length 
of 220 nm and was also screened for LTA by PHA (-H-H-) using a single 
dilution sample. In addition column fractions were tested for the pre- 
sence of chloride ions by placing one drop of a saturated AgNO solution 
on a coverslip containing one drop from each test tube. Any resulting 
precipitation was evaluated on a +1 to +5 basis and plotted accordingly. 
It was empirically determined that not only CI reacted with the AgNO 
resulting in insoluble AgCl, but the NaN and tris carbonate in the 
buffers reacted as well. The presence of propanol was monitored indi- 
rectly by changes in test tube volume. Since LTA, azide, and tris 
carbonate all absorb at a wave length of 220 nm the combination of ultra- 
violet light screening, the AgNO. precipitation test, and visual inspec- 
tion of volume changes per test tube proved to be invaluable for rapidly 
discerning the location and separation of LTA from contaminating salts 
and solvents. The entire contents of peak I were pooled, frozen, and 
lyophilizod. The final product was referred to as LTAosx (extracellu- 
lar lipoteichoic acid purified by Octvl Sopharose hydrophobic affinity 
gel chromatography). The typical mass yield from stub a procedure 
was about 60-70°. Percent recovery <'( LTA at various points in the 
procedure is summarized in Table h. 



Figure 14. Simultaneous removal of salt and propane) 1 free LTAosx 

by LH-20 gel chromatography. Symbols: (•) A ?? absor- 
bance; (o) Volume/ test tube: (+) Ant igen icity~as deter- 
mined by PHA; (Shaded Area) relative degree of precipi- 
tation of salt and other low molecular weight materials 
as determined by AgNO test. 



3ani is3i / ai/vmoA 



69 




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Phosphatidyl choline vesicle (PVC) purification of LTA using U r: 



labelled p hosphatide; I gjloUne. Approximately 5 X LO* 



14 
DPM of C 



labelled phosphatidyl choline were added to 40 mg of phosphatidyl 
choline dipalmitoyl. Phosphatidyl choline vesicles (PVC) were pre- 
pared as described in Materials and Methods. Three test tubes contain- 
ing identical volumes and concentrations of non-labelled PGV were pre- 
pared simultaneously and 2.0 ml of LTAppx (1.5 mg/ral) were added to each 
test tube. Fifty microliter samples from the C containing test tube 
were removed at various steps during the purification process and an- 
alyzed as described (Materials and Methods). A standard chloroform 
quench curve was constructed and all reported counts represent corrected 
DPM values. Table 5 depicts the distribution of 4 C counts at various 
steps in the purification procedure. Utilizing this procedure as des- 
cribed, essentially no contaminating phospholipid could be detected in 
the final product. The typical mass yield of product: via PVC purifica- 
tion was about 10-15%. Percent recovery of LTA at various steps in the 
procedure is summarized in Table 6. 

Comparison and summar y of LTApcx vers us L TAo sx . As indicated in 
Table 7, both methods of LTA purification removed the majority of pro- 
tein as compared to the total, amount abai Table in the LTAppx. Both, 
methods ostensibly recovered > 80% of the original LTA. However, the 
major difference between the two products is reflected in the percent 
total, mass recovery and the concomitant Increase in percent carbohydrate 
in the final LTAosx product. This latter difference can be most readilv 
discerned by observing the composite gas ehromar ograph tracings in 
Figure 15. The carbohydrate standard (CHO-STD) depicts the typical 
ehromatograph of glucose and galactose after preparing tr Imothvlfii lv 1 





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of t.TA containitiR preparations 

iv . Abbrev in t ions : (HAN) 



Mann i t ol 



Figure 15. Carbohydrate analysis 

by gas liquid chroinatograp 

Mannitol; (GLC) Glucose; (GAL) Galactose? 

was incorporated as an internal standard with alt 

samples . 



76 



L T A pp> 



MAN 




/ / 



ester (IMS) derivatives as described in Materia! and Methods. An inter- 
nal mannitol standard is included with all samples. The LiAppx chroma- 
tograph represents the typical carbohydrate profile achieved with 
partially purified LTA. The tracings for LTAosx and LTAppx contrast 
the qualitative and quantitative differences in carbohydrate content. 
The second two chromatograms,deacylated cardiolipin (f, p ) ] and eardio- 
lipin, were included as a comparison of how a naked polyglycerol phos- 
phate backbone might be expected to react under the described conditions. 
The base line instability of the C.^ looks remarkably similar to the 
profile of the purified LTApcx, The procedure for purifying deacylated 
cardiolipin requires passage through Sephadex columns. it is quite con- 
ceivable that the minute quantities of unidentified carbohydrates which 
are indicated may be due to dextran contamination from the column. 
However, it would be difficult to account for the same source of contam- 
ination for the LTApcx since gel chromatography was not used in the final 
purification. On the other hand, the similarity of the indicated ehroma- 
togram tracings may he more than mere coincidence and may reflect actual 
reactions of the derivntizing agent with the polyglycerol phosphate 
backbone. This latter hypothesis is supported by the fact that an 
unidentified trailing "carbohydrate" peak oi significant mass appears 
in both the cardiolipin and C P, chromatography. The Kf value of this 
peak is similar (hut vet suspiciously disparate) to the retention tine 
normally observed for H-gIuco.se. However, if indeed this peak does 
represent B-glucose, one is hard pressed to rationalise why a corres- 
ponding .-.-glucose peak does not occur as well. In either ca.se, it is 



Deacylated cardiolipin was prepared by the method of Wilkinson ({•>')) 
and was kindly provided by R. Craig, University o( Florida. 



7:< 



apparent that the LTAosx still contains «j t«n if icnnt amounts of carbo- 
hydrate contamination in contrast to the lower yield, but highly puri- 
fied LTApcx. 

A summary of specific activities relative to PllA activity is pre- 
sented in Table 3. 

Inh ibition of comp le ment me diated l_-/sjs of LTApc x treated EA. 
Once a highly purified preparation of LTA was obtained, it was necessary 
to confirm the results that had been previously established with LTAcx, 
As can be seen in Figure 16, not only were LTApcx treated EA refractory 
to complement mediated lysis, but in addition the general profile was 
remarkably similar to LTAcx treated EA. As indicated, LTApcx was used 
in concentrations five-fold to ten-fold less than those used with LTAcx 
to achieve comparable degrees of inhibition. PHA titers typically indi- 
cated LTA saturation of the cells. 

Mi. c lLL..i^_JiTAp_o^jDiT_j^lie .ly sis of various cel lular complement 
componen t intermediat es. Sheep E, EA , and various complement inter- 
mediates were treated with LTApcx (100 us/ml in DVB) and washed exten- 
sively in DGVB. Percent Lysis and inhibition of OH units were deter- 

50 

mined as described in Materials and Methods. The results of these 
experiments are summarized in figure 17. As with LTAcx treated cells, 
E, EA, and EAC.1 were all refractory to lysis by complement. Likewise, 
EAC142 and EACi-7 intermediates were totally unaffected by the presence 
of LTApcx on their cell surface, the on 1 v observable difference in the 
activity of LTApcx on cellular intermediates versus LTAcx was that LACK 
cells were inhibited to a lesser degree with LTApcx than LTAcx. 

Eff e ct of LTAcx and LTApcx _on flu id p hase C I . As previously 

discussed, 01 is not required for lysis once SAC 1 42 are formed but it. is 



79 



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Passive hemagglutination (P!iA) titration and inhibition 

of complement mediated lysis of FA treated with varying 
concentrations of LTApcx. 



81 





% PHA 

-50 % PHA 

I 00 % PHA 



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LTAcx (/zg/ml) USED IN EAlta P c* PREPARATION 



Figure 17. Effect of LTApcx on the complement mediated lysis of 
various cellular complement component: intermediates. 



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84 



essential until that point is reached (148). In addition, many poly- 
anionic substances are known to directly affect CI bv interfering with 
Clq binding or CI esterase (('Is) activity (39,40). Because LTA is 
polyanionic due to the polyglycernl phosphate backbone and because 
cellular intermediates beyond the EAC142 step were no Longer inhibited, 
it seemed reasonable to hypothesize that LTA was behaving like a poly- 
anion and directly affecting CI. 

To test this hypothesis, LTAcx and LTApcx were preincubated with 
functionally purified human CI at 30° / L ^ minutes. Residual CI activity 
was titrated as described by Rapp and Borsos (139) and activity was com- 
pared against buffer treated controls. The results shown in Figure 18 
indicate that although LTAcx consumed CI activity, purified LTAncx did 
not. 

Purifica tion of hu man Clq, Cls and (l is. In an attempt to further 
eludicate a possible sice and mechanism of C inhibition, human CI sub- 
components were purified by the methods .aid mod if teat ions previously 
described (Materials and Methods). Although homogeneity beyond func- 
tional purity was not essential, the methods employed yielded highly 
purified products. Figure 19 demonstrates Clq homogeneity by immuno- 
diffusion against several monospecific antisera. Precipitation bands 
of identify were observed in adjacent wells containing the whole human 
serum starting material, the purified Clq final product, and a highly 
enriched Clq prior to final precipitation (Figure 19, plate 5, well 
numbers A, C, and E) . As can be observed in Figure 20, disc gel electro 
phoresis of the final product revealed a single dark staining band which 
barely migrated into the separation gel. These, results are consistent 
with the observations of other investigators (144). 



Figure 18. Effect_of LTAcx and LTApcx on functionally purified 
human CI. The upper graph represents a residual el 
titration after incubation with LTAcx (500 up/ml). 
The lower graph represents the results from an analo- 
gous experiment using LTApcx (500 ug/ml) instead of 
LTApcx in the incubation mixture. Symbols: (.•) CI 
incubated with buffer; (o) CI incubated with the 
appropriate LI'A containing extract. 



$f> 




16,000 8,000 4,000 2,000 

RECIPROCAL OF HUMAN CI DILUTION 



Figure 19. Immunodiffusion and precipitation analysis of various 
steps in the purification of human 01 q. Purification 
was achieved by repeated fractional precipitations of 
whole human sera in buffers varying in ionic strength, 
pH, and concentrations EGTA or ED TA (144). 
Well designations: 

(A) Whole human sera (starting material); 

(B) Supernate from first precipitation; 

(C) Supernate from second precipitation; 

(D) Supernate from third precipitation; 

(E) Material from resuspended pellet prior to 
final precipitation. 

(F) Final product (purified Clq). 

Plate designation: (1) Center well contains nnti-lgO; 
(2) Center veil, contains Anti-IgA; (.3) Center well 
contains anti-whole human sera: (A) Center well 
contains anti-IgM: (5) Center well contains anti-Clq. 



88 




Figure 20. Disc gel electrophoresis of purified human Clq 
Cathode was at the top. 



90 




The procedures for Cls and Cls purification were modified only in 
that an Ionic gradient was used in the final purification step of both 
reagents rather than the stepwise elutlon utilized by Sakai and Stroud 
(35). The rationalization for this modification was that a difference 
in binding capacities of the DEAE matrix could have deleter iously 
effected the eiution characteristics of the Cls (Cls) at a fixed ionic 
strength. The eiution profile of Cls is shown in Figure 21. Tt should 
be noted that two peaks of material which .absorbed light at a wave length 
of 280 nm were resolved during the gradient eiution. Both peak II and 
and peak III reacted with monospecific ant isera to Cls, however, only 
peak III contained Cls activity. Peak II presumably represents an in- 
active form of either Cls or Cls. No such extraneous peak was resolved 
during DEAE chromatography of Cls. 

Immunoelectrophoretic analysis of purified human Cls and Cls on 
17. Noble Agar is depicted in Figure 22. Results indicate a difference 
in electrophoretic mobility of Cls and Cls which is consistent with the 
observations of previous investigators (33). Also, there was a "gull 
wing pattern displayed by Cls apparently representing mi crohe tero- 
geneity of the activated procst erase . This too has been observed by 
previous investigators (33). 

Effe ct of LT Apcx jm. the jibj 1 i_ty of C Is to consume C4 a_n_ d__C 2_ _a ct tvitv . 
As previously discussed, activated CI. esterase (Cls) is capable of 
cleaving C4 into C-'ta and C4b (43) as well as cleaving ('2 into C2a and 
C2b (46). In either case, the active fragments rapidly decay and if not 
quickly attached to membrane sites, lose their ability to do so. Iho 
ephemeral nature of these active fragments can he \t^cd as sensitive in- 
dices of Cls activity. As described in Materials and Methods, emial 



Figure 21. DEAE eiution profile of human Cls. Peak [ contains 

CJ s activating proteins (functionally pure Clr); Peak 
II contains nonfunctional CLs; Peak III contains 
functional, non-activated Cls. Symbols: (•) Absor- 
bance at A (maximal absorbance for most proteins; 
(o) Relative salt concentration (RSC) as measured 
by electroconductivity . Arrows indicate the addition 
of high ionic strength Sodium Chloride buffer. 



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gure 22 



Immunoelo C (- r _ r . 

contained purif ^fjfl P«lf led Cl s; wel Tj 
-holehu man sera _ «•• « 1C contained 
" s CCl.)j tro hs r fl contained . ntl 

ant i Cls (cIT) lG human sera and 257 



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96 



volumes of CIS, functional I v pure CA or Cl, and LTApcx were mixed and 
incubated at 37°/5 minutes. The incubation mixture was then serially 
diluted in DGVB, and the residual titers of C '. or C2 were determined 
and compared against a buffer treated control. As shown in. Table 9. no 
appreciable difference in Cls activity can be observed when even 200 us/ml 
of LTApcx were used. Also note that LTApcx preincubated with either C4 
(Expt. Group 2d) had no significant effect on residual activity. 

Effect of LTApcx on the ability cf Cls to hydrolyze TA rle . The 
ability of Cls to hydrolyze the synthetic substrate TAMe is another L«de:< 
of Cl"s activity. As can be seen in Figure 23, essentially no inhibition 
of Cls activity was observed when Cls is preincubated with LTApcx and 
TAMe. 

Eff ect of LTA pcx on the ability of Clq to bind to target cells . 
Since Clq is the recognition unit of the classical pathway of complement, 
any alternation in its ability to react with the antigen-antibody com- 
plexes on the surface of EA would have profound affects on the ability 
of complement to lyse those cells. Therefore, equal volumes of puri- 
fied human Clq and LTApcx (10 Ug/ml) were preincubated at 30*715 minutes. 
After preincubation, Clq was serially diluted in IKA'R and Clr and Cls 
reagents were added. Hemolysis was then developed as described (Materi- 
als and Methods). Again there was no apparent inhibition (results not- 
shown) of activity. The major criticism of this experiment is that, the 
LTApcx concentration used to preineubate with Clq is 10-fold less than 
what was normally used in fluid phase tnae 1 1 vat ions. The reason for the 
use of this lower concentration was to insure that the LTA would be 
sufficiently diluted at the time of LA addition. If significant amounts 
of LTA were present in the incubation mixture, EA f;rA would fern, thus 
generating a false positive inhibition duo to the refractory nature o( 



97 



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Figure 23. Effect of LTApcx on the ability of Cts to hydro Uze 

TAMe. As the synthetic substrate TAMe is hydrolized', 
there is an Increase in A.,,, absorbing material. in 
this experiment, Cls and TAMe were incubated together 
in the presence of LTApcx (100 pg/nll at room temper- 
ature (24°C). Symbols: (>) Cls and TAMe phis buffer: 
(o) Cls and TAMe plus LTApcx. 



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EA . Despite the lower concentration, previous data with LTA treated 

LTA 
EA (Figure 16) indicated that even at this concentration, inhibition 

should have been significant if indeed Clq were the site of inhibition. 

8 
Instead of this anticipated inhibition, 1.36 X 10 SFU/ml of Clq were re- 

8 
covered from the incubation mixture originally containing 1.50 X 10 SFU 

Clq/ml. Due to assay variation this difference was considered insignificant 

Effect of LTApcx on CI transfer . In another attempt to elucidate 
the effect of LTA on the Cl molecule, the interference of the normal 
ability of Cl to transfer from cell to cell tinder conditions of high 
ionicity was investigated. Two different types of transfer tests were 
performed. In type I, EA were treated with LTA and then cT was added. 
In type II, EAC1 were prepared and then LTA was added. Not so sur- 
prisingly there was no inhibition of Cl transfer as measured by 'Hemo- 
lysis of EAC4 cells. However, there was an i ncreas e in the Cl trans- 
ferability of cells containing LTA. As can he seen in Table 10, this 
phenomenon was repeatable and was observed in both types of experiments. 

Differences in complement mediated lyti( : susceptability of LTAcx 
treated EAC4 ver su s EAC14 . Buffer or LTAcx (250 ug/ml) was used to 
treat EAC4 using procedures described in Materials and Methods. Various 
limiting concentrations of human d were then added to aliquots of the 
cells and lysis was developed as previously described. Alternately, 
EAC14 were prepared using various limiting concentrations of human Cl . 
Aliquots of cells were then treated with LTAcx (250 tig/ml) or with buf- 
fer. After the cells were washed extensively in buffer, lysis was 
developed as previously described. As shown in Figure 24, EAC14 treated 
with LTA are considerably more refractory to complement mediated lysis 

than are EAC4 treated with Cl. 
LTA 



TABLE 10 



_ Comparison of the Relative Numbers of Effective 
CI Molecules Capable of Transfer from EAC1 Treated with LTApcx 



10L 



Sample 



Experiment 
Number 



Effective Number cf CI 
Mol ecu! es Transferred/Cell 



EAC1 



LTA 



EAC1 



LTA 



EAC1 



LTA 



1 
2 
3 



175 
124 
153 



EAC1 



EAC1 



DVB 



DVB 



137 

115 



EA LTA CT 
EA LTA Cl 



18 5 

178 
215 



EA DVB C1 d 
EA DVB CT 



132 
182 



EAC1 vere generate/.! and treated with LTApcx at 1 00 up/ml in DVB. 
After extensive washing, the CI capable of transfer was titrated. 

Control EAC1 treated with DVB for }0"/l.5 minutes 

F.A . , were prepared (100 Jig LTApex/ml) and afttT_thc cells were 
extensively washed F.A CI were generated. The CI capable of 
transfer was then titrated. 



Control i'AGl treated with DVB while In the FA state. EAC1 pre- 
paration and CI transfer exactly paralleled the LTA treated cells 



Figure 24. Differences in complement merlin ted lytic SUPC.optahiU.tv 
of LTAcx treated EAC4 versus FAC14. Upper graph: RAC4 
were treated with buffer or with LTAcx (250 ug/ml). 
Various limiting dilutions of human CI were then added 
to aliquots of the cells and Lysis was developed according 
to procedures described in Materials and Methods. Lower 
graph: [SAG14 were prepared with various limiting dilu- 
tions of CI. Aliquots of cells were then treated with 
LTAcx (250 iig/tnl) or with buffer. After extensive 
washing, lysis was developed according to procedures 
described in Materials and Methods. Symbols: (•) 
Buffer treated cells; (o) LTAcx treated cells. 



103 




8,000 4,000 

RECIPROCAL OF HUMAN C 



2,000 
DILUTION 



L0-'< 



In addition to the above mentioned experiments, several other 
assays to elucidate the mechanism of inhibition were attempted. Unfor- 
tunately none of these experiments led to results that were consistent 
with any models attempting to explain how some complement cellular 
intermediates became refractory to complement lysis when pretreated 
with LTA. These experiments and their summarized data are presented 
below: 

CI uptake by EA . EA were prepared with LTApcx at a con- 
LTA LTA 
centration of 100 iig/ml using procedures described in the Materials 

and Methods. Buffer treated EA were also prepared at the same time. 

10 
Human Cl (approximately 1.0 X 10 SFU/ml) was reacted with allquots 

from each cell preparation and incubated for 15 minutes at 30°C. The 

cells were pelleted by r.entrif ugation and the supernates analyzed for 

9 
residual Cl activity. Approximately 6.5 X 10 SFU Cl/ml remained in the 

9 
supernate of the buffer treated controls whereas approximately 6.8 X 10 

SFU Cl/ml were titrated in the supernate of the EA treated cells. 

LTA 
Because values fluctuated by 5-8 % from one experiment to the next, this 

slight degree of enhancement was not considered significant. 
HIT HU 

M£J^lLiifL_.li^l!Ll^ n ^l f _ t( -- r preincubation of CA _ with EAC1 

9 '"" LTA 

Human C4 (approximately 4.0 X 10 SFU/ml) was added in equal volumes to 

EAC1 which had been preincubated with cither LTApcx (100 |ig/ml) or with 
buffer. The mixture was incubated at 30° for 15 minutes and residual (". 
activity was titrated as described in the Materials and Methods. EA 
were incubated with the CA reagent as a negative control. Results in- 
dicated that there was approximately a 30% decrease in residual C'» 



titer of the supernates previously incubated with EACl versus the negative 

control which consisted of C4 incubated with EA. However, both EACl 

— LTA 

and EACl consumed identical amounts of C4 (residual supernate C4 

buffer 9 9 

activity was 2.71 X 10 SFU/ml and 2.79 X 10 SFU/ml respective.lv). There- 
fore, it was concluded that LTA had no apparent effect on C4 uptake by 

EACl. 

GP 
R esid ua l_Cj__ti^at_io_n af ter preincubat ionj^Uh_EACl4 . Guinea 

10 ~ ' lta 

pig C2 (approximately 1.5 X 10 SFU/ml) was added in equal volumes to 

EAC14 which had been preincubated with either LTApcx (.1.00 ug/ml) or 
with buffer. The mixture was incubated at 30° for 12 minutes and resi- 
dual C2 activity was titrated as described in Materials and Methods. EA 
were incubated with the C2 as a negative control. Results Indicated that 



9 
approximately 35% (5.3 X 10 SFU C2/mi) of the available C2 was utilized 

o 

by the EAC14 complexes and approximately 29?: (4.4 X 10 SFU C2/ml) were 

utilized by the EACl 4 complexes. Despite the fact that the supernate 

LTA 
from the C2 incubated with EACl 4 had slightly more residual C2 activity 

LTA 
(approximately 71% of the C2 activity still remained in the supernate 

after incubation with EACU ), a difference of only 6% i s within cx- 

LTA 
peridental variance of this assay. Therefore, it was concluded that LTA 

had no apparent effect on C2 uptake by EACI4. 

Inhibition of lysis of EA by LTA from ot her bacterial sources . 

Additional evidence indicating that LTA might be primarily responsible 

for the C inhibition phenomenon came from hemolytic assays utilising 

LTA from other sources. Dr. R. Doyle (Hept. o( Microbiol ogy and 



106 



Immunology, Univ. of Louisville) provided samples of 1/i'A purified from 
Bacillus, suhtllls strain gta B'290. Purified [,TA from Lact obacillus 
casei ATf:( < 7<i6q was obtained from the Institute of Dental Research, 
Sydney, Australia, and Dr. A. S. Bleivels (Dope of Microbiology and 
Ceil Science, Univ. of Florida) provided a sample of partial iy purified 
LTA from Streptococcus mu tans strain AHT. Each preparation was mixed 
with EA; the cells were thoroughly washed and analyzed using the pre- 
viously described techniques of PHA and suscept lb i 1 itv to whole comple- 
ment lysis. As depicted in Table 11, all preparations contained material 
that reacted with anti-LTA by PHA and all such cells— especially those 
prepared with the purified I. easel— were more resistant to the hemo- 
lytic action of complement: than were untreated controls. 



TABLE 11 



Percent Inhibition and PHA Titer of F.As 
Treated with LTA Containing Extracts from Several Sources 



h c 

Source of LTA Percent Inhibition PHA 



S_. mutan s BHT 40 

S. mutans AHT 35 



1600 
1600 



L. casei (ATCC 7469) 75 3200 



,. subtilis (gta B290) 70 



1 M')0 



a The LTA extracts from all sources were used at a concentration of 
50 pg/ml. in VBS. 

b EAs were treated with the appropriate LTA-extrac! and hemolysis 
wa 
of h 
units . 



developed by incubation of the cells with several dilutions 
uman C (37°/60 minutes). Values represent inhibition of CH 5Q 



C PHA titers are expressed as the reciprocal of. the final dilution 
of specific anti-LTA which caused hemagglutination. 



DISCUSSION 

Evidence has been provided for the Inhibition of complement 
mediated lysis of target cells by an extracellular material obtained 
from Streptococcus mutans BHT. This material has been identified .1 ; 
lipoteiehoic acid (LTA) and is a plasma membrane constituent of most 
gram positive bacteria (107,108). Various gram positive bacteria 
isolated from the oral cavity differ in the amount of LTA they excrete 
into the external environment. S. muta ns BHT is an example of a endo- 
genic streptococcus that not only produces copious amounts o£ LTA (1,20) 
but its ubiquitous nature provides for a constant, inundation of LTA 
and other metabolites into the gingiva] crevices of the oral cavity. 
The presence of a complement reactive component iii the microenvi ron- 
ment of the gingival crevices could result in any number of biological 
effects. Direct activation of the complement system (either classical 
or alternative) may result in the destruction of nearby "innocent by- 
stander" cells. This is particularly true if the activator is cvto- 
philic and thus capable of "sensitizing" nearby host cells. Activation 
of complement in the gingival crevices can also result: in osto.oelast- 
mediatcd bone resorbtion (14). This phenomenon is further complicated 
by the fact LTA and LI'S (and ostensibly hvdrid mieells of the two) are 



1 Some bacteria are known to lack LTA in (heir membranes but. in these 
cases "LTA- like" molecules arc inserted instead. Lxampl.es are the 
iipomarman of Micr ococcus lysodeikt icus (150) and the F-antigen of 
D ip iococcus pneumoniae (115). 



108 



10? 



also capable of stimulating osteoclast mediated bone resorbtion (17). 
Even without profound activation of complement, the possession and 
release of complement inhibitory substances -night confer a certain 
degree of survival value on the organisms producing them. Thus In 
the face of immunological challenge, the complement system nay be 
blocked from reacting against the bacteria producing such factors. 
It may be more than coincidence that gram positive organisms such as 
Micrococcus lysodeik t icus lacking LTA in their cell membranes are also 
susceptable to lysis by the synergism of lysozyme and complement (151). 
All other gram positives containing intact LTA in their membranes are 
notoriously resistant to complement lysis even in the presence of 
lysozyme (151) . 

Three lines of evidence have been obtained which suggest that 
the active inhibitory factor is 1 ipteiciioic acid (LTA). The inhibitor 
co-purified with LTA when extracellular material from spent culture 
was fractionated by gel-filtration and was purified by adsorbtion to 
phospholipid vesicles. Sheep erythrocytes which had been treated with 
§.- nutans BUT extracellular extract became resistant to lysis by com- 
plement and they also became coated with LTA as judged by PHA using 
antibodies monospecific for purified LTA. The amount of LIA present 
on the cells paralleled the degree of lytic resistance that was 
acquired by the treatment. Purified LTA and LTA- rich fractions from 
other bacteria also caused sheep erythrocytes to become resistant to 
complement mediated hemolysis. Again, PITA assays indicated that cells 

which became resistant to lysis had LTA on their surfaces. 

Experiments using crude extracellular LIA (LTAcx) provided evi- 
dence for the consumption of whole human complement activity. When 



1.0 



preincubated with various concentrations of LTA, whole human sera lost 
complement activity '.n a dose-dependent fashion. Individual component 
titrations revealed that not only C3, but the early components CI, Ci, 
and C2 were consumed to some degree. However, no C3 consumption was 
observed if the preincubation was performed with isolated C3 or in the 
presence of EDTA., If EGTA-Mg were substituted as the chelating agent, 
only a minimal restoration of C3 consuming activity was observed. 
These results indicated that not only were calcium and magnesium ions 
necessary for the anti-complementary activity, but there was a require- 
ment for some factor(s> in whole sera as well. This "factor" is most 
likely natural antibody directed at LTA or some component of the crude 
extract. This resulted in the formation of a typical antigen-antibody 
complex with subsequent classical complement consumption. 

Experiments using sheep erythrocytes in various stages of com- 
plement component fixation provided evidence that LTA was not only 
capable of spontaneously adsorbing to the surface of these cells, but 
also rendered many of the intermediates refractory to lysis. When 
sheep red blood cells, EA, or EACl were treated with MA, all became 
resistant to complement lysis. Lipote icho i c acid treated F.AC U were 
somewhat less resistant to lysis and all cellular complement inter- 
mediates beyond EAC14 were no longer protected. 

Conversion of colls to hemolytic resistance by treatment with 
LTAcx can aid in the interpretation of the C2 consumption data depicted 
in Figure 4. As indicated, the degree of (!2 consumption was dispropor- 
tionate compared to loss of CI and C '. netivitv. However, the commer- 
cially available human C2 used in these studies had a fairly low titer. 
As a result, the dilutions made after the preincubation step were not 
sufficient to prevent substantial amounts of the LTA from binding to 



11 



the cells and being expressed Ln the C2 titration. What anpeared to 
be consumption of C2 activity was actuallv the Inability of the comple- 
ment system to lyse resistant cells. Because of the greater extent of 
dilution, the same phenomenon did not influence CI, 04 and C 3 titrations. 



Because the EAC142 and EAC1423.'367 intermediates were not effected 
by LTA, some component no longer necessary for their stability was a 
likely site of inhibition. CA was probably not the site of attack 
since this component is a necessary part of the C'3 eonvertase (152), 
and EAC142 were not inhibited. Only CI is expendable after the EAC142 
complex is formed and thus CI seemed to be the most likely candidate 
for the site of inhibition. 

The first consideration was the possibility that LTA was causing 
inhibition of complement mediated lysis by blocking fixation of el to 
antibodies specific for sheep, erythrocytes or by Mocking the site of 
antibody attachment. The fact that the inhibitor functioned equal!, 
well when it was presented either before or after the addition of speci- 
fic antibodies to the cells indicated that blockage of antigenic sites 
was not the mechanism of inhibition. Tin's experiment did , 1oC ru!o out 
the possibility that the inhibitory substance could react with (he {'.]' 
fixation sites on immunoglobulin molecules. However, Figure 10 shows 
that preincubation of LTAcx with anti-sheep I., hemolysins did not 
decrease the hemolytic antibody titer of the serum. [f LTA were capable 
of binding or inactivating immunoglobulin molecules (either specifically 
or non-specif ically) then the titer of the antiserum should have been 
reduced as a result of treatment with the bacterial extract. 

There was some speculation that LTA might inhibit complement medi- 
ated lysis by inducing some alteration in the structure of the target 



col] membrane. However, one would expert ,11 „f the complement com- 
ponent intermediate cellular complexes to he equally affected hy I.iA, 
when in actuality this was not the ease. Lt is possible that some of 
the complement components could block the attachment of the inhibitor 
to cell membranes so that the material would have no opportunity to 
cause membrane alteration. This is an unlikely possibility because 
even EAC 1421567 had LTA on their surfaces. 

The highly purified LTA necessary for the final site of action 
and mechanism studies proved to be considerably more difficult to ob- 
tain than previously anticipated. As suggested by the results in 
Figures 13 and 15, and Tables U and 7, the Octyl Sepharose method of 
LTA purification did not sufficiently resolve the LTA from tenacious 
polysaccharide contamination. This method yielded almost quantitative 
recovery of LTA (as determined by PlIAg) and a Lso a significant portion 
of the total mass which was applied to the column. However, considering 
the contaminated nature of the final product even when an elation gra- 
dient was utilized, it was determined that a significant percentage of 
the mass was probably polysaccharide. Indeed, gas Liquid chromatography 
of extracellular LTA purified by Octyl Sepharose (LTAosx) indicated 
that as much as 30% of the final weight was carbohydrate, presumably 
existing as polysaccharide (Figure 1.5, Tabic 7). 

In contrast, the somewhat more elaborate method o\ purifying LTA 
by adsorbtion to phosphatidyl choline vesicles (PCV) yielded a product 
that was virtually devoid of all nucleic acid, protein, and carbohy- 
drate contamination (Figure 13. Tables 5,6, and 7). (able 5 indicates 
that although approximately 11 of the radioactive ' ''c used to label the 
PCV was lost during washing procedures (and ostensibly, a percentage 



ti: 



of bound LTA as well), over 92% of the label could be accounted for in 
the chloroform/methanol filtrate and the first filter washing. Only 
O.OOhZ of the label was present in the final product therefore, elimin- 
ating phosphatidyl choline as a source of contamination. Figure 15 and 
Table 7 indicate that less than 5% polysaccharide, contamination can be 
detected in the final product by gas liquid chromatography. Considering 
the unusual profiles obtained from the gas liquid chromatography of 
both cardiolipin and deacylated cardiolipin (Figure IS), u is likely 
that the percentage of contaminating polysaccharide in the final LTApcx 
preparation is even Less than 51. As can be seen in Table 6, approxi- 
mately 85% of the LTA in the original partially purified extract can be 
accounted for in the final product and washings. However, it should be 
cautioned that the method used for these determinations (PHAg) is semi- 
quantitative at best and is only considered accurate to within one two- 
fold dilution. 

Although the percent protein of all partially purified samples 
was determined by amino acid analysis, unfortunate Lv the tremendous 
quantity of purified material required in analysis for < 5% sensitivity 
in analysis, exceeded the total amount of purified material available. 
In fact after allocating fixed quantities of purified product for the 
various other quantitative and complement assays, the required 5-6 mg 
of purified LTA needed for amino acid analysis far exceeded the poten- 
tial amount available from the LTAppx. For this reason, the Bio-Rad 
Protein Assay was used to estimate the total amount protein in each 
sample. As can be seen in Table 7, there was a relatively close cor- 
relation between values determined by amino acid analysis and those 
determined using the liio-Rad Assay. It is therefore reasonable to 



IU 



assume that the values given for the final products are at least a 
close indication of the total percent protein available in each product. 
Although the values may seem high, it should he remembered that (1) the 
total amount of protein available in the sample represents a Lower limit 
for the accuracy of the assay, and (2) the standard protein curve 
(human albumin) used to convert optical density readings to ug of pro- 
tein may not accurately correlate the reactions of the limited number 
of amino acid residues available in the final product. Attempts to 
verify these values with the biuret reaction (153) and the I.owry Pro- 
tein Assay (154) were unsuccessful. Biuret was too insensitive whereas 
the Lowry proved to be unreliable due to its reaction with glycerol to 
give a false positive reaction. Despite this shortcoming , all other 
factors indicate that LTApcx represents the most highly purified LTA 
from S. mutatis BHT that any laboratory has yet achieved. 

The results from the final experiments to determine the site and 
mechanism of complement inhibition by I.TA were equivocal. Like the 
purification of LTA, establishing the site and mechanism of inhibition 
proved to be considerably more challenging than anticipated. Prelimin- 
ary data utilizing LTAcx quite consistently suggested that CI was the 
site of action and interference with binding affinity (Clq dysfunction) 
or with esterase activity (Cls dysfunction) was the mechanism. These 

conclusions were based on the fact that FACIA but not EAcJ~42 were 

l.TA ' "LTA 

inhibited and also that the titer of fluid phase Cl preincuhated with 
LTAcx was s i gn i f ican t 1 v reduced. Considering the pnlvanionlc nature of 
LTA conferred bv the polar po 1 yg lyecro 1 phosphate backbone, it appeared 
that LTA represented a model system for polvanionic interference of C\ 
function. Such activity has been ascribed to dextran sulfate polyvinyl 



11 



sulfate, heparin, poLviosinic acid, chondroitin and many other poly- 
anionic compounds (39,40) in addition to DMA. RNA (155,156), and 
carrageenin (157). [t was dismaying to find thai although LTApc* 
maintained anti-complementary activity with the appropriate cellular 
intermediates, all f luld phase inhibition of C J ^ nhrop;Ued (FlRurM 
17 and IS). All subsequent experiments attempting tn define Clq, C 1 s . 
or Cls dysfunction were negative. The only experiments that gave sug- 
gestive results were the d transfer assavs. Even he re , lnstend of thc 
anticipated inhibition of CI transfer, over 20% enhancement of transfer 
was observed (Table 10). Thus, in light of these data obtained with 
purified LTA it was necessary to devise new molecular models to exnlain 
the mechanism of lytic inhibition of certain complement intermediates 
by highly purified LTA. Some possibilities are discussed below: 

1). Attachment of LTA stericailv blocks the affixation of Ci to the 
Fc portion of the immunoglobulin. Thus. If CI does not attach properly, 

or is prevented from nttnehino it- 1 1 i .i i 

•'(lacin.ng .it all, the complement cascade will never 

be initiated. 

2). Although Cls" activity was not effected fluid phase, perhaps such 
activity would be abrogated once the CI molecule became associated with 
the cell membrane. [f so, EAC1 would no longer be capable of hvdrolizing 
C4 or C2 again, the cascade would be. terminated . 

3). LFA directly interacts with fluid phase Ci or ('2 thus preventing 
them from combining with the appropriate sites on the membrane. 

4). The attachment of LTA leads to increased fluidity of the mem- 
brane resulting in the displacement o^ loeselv attached molecules. If 
some of these less tenacious molecules include any of the early comple- 
ment components, the physical loss of these components would terminate 
the lytic attack sequence. 



1 1 ,, 



5). The attachment of LTA to the cell membrane prevents the subse- 
quent attachment of the C4 or C2 active fragments (i.e. Mb or C2n 
respectively). Thus, the activities of all complement components 
would remain intact and no observable dysfunction should be observed. 
However, if C4b or C2a were in the least impeded in their attachment 
to the cell membrane, these active fragments would rapidly decay and 
lose their ability to do so. 

If the first model accurately portrayed the mechanism of inhibi- 
tion, one would predict a decrease in CI uptake by EA . This predic- 

LTA ' 

tion was not corroborated by experimental results. In addition, this 

model would not explain the high degree of inhibition of cells in the 

EAC1 state where CI is already attached. 

If the second model were true, one would predict a decreased 

consumption of fluid phase C4 or C2 after pre incubation with EACl , . 

L PA 

Again, such was not the case. Neither residual C't activity when in- 
cubated with EAC 1 nor residual C2 activity when incubated with 
EAC1~4LTA WaS a PP reci< ' ihl y different from their buffer treated controls. 

Model three would predict a decrease in fluid phase activity of 
C4 or C2 when preincubated with LTA. As demonstrated in Table 8, no 
such decrease in activity was observed. 

Model, four maintains (hat the attachment of LTA would somehow 
alter the membrane such that loosely attached components would be 
released more readily. The first: problem with tin's model is that the 
attachment of the early complement components to the membrane is quite 
tenacious. In fact, some evidence suggests that membrane attachment 
of cytophillis C4b is accompanied by the formation of eovalent bonds 
(158,150) . Once attached, it seems unlikely that C4b would be readilv 



117 



released. CI Is not attached to the membrane at all, but rather is 
combined with the Fc region of the hemolysin antibody. Therefore, this 
model would predict that either CI is released from the antigen-anti- 
body complex (very much akin to the predictions and shortcomings of 
model one) or that the entire antibody-Cl complex is reJ eased from the 
cell membrane (with or without the accompanying antigen). Such a 
mechanism is somewhat exotic, but not totally improbable. Recent evi- 
dence suggests that the binding of serum albumin, immunoglobulins, or 
complement can effect a release of phospholipids from liposomes (160, 
161,162). Perhaps the attachment of LTA can likewise evoke such a 
release of cell membrane constituents and in the process, release the 
immune complexes as well. Experiments utilizing f 131 labelled hemolysin 
antibody would demonstrate, whether the antibody was maintained on the 

i "5 I 

cell surface or released into the medium. Likewise. I labelled CI 
could be used to determine if CI were released. 

Of all the proposed models, number five most likely portrays the 
actual mechanism of inhibition. This model asserts that the affixation 
of LTA to the surface of the cell delays or prevents the rapid associa- 
tion of C4b (or C2a) with its respective site on the cell membrane. As 
previously discussed, once C4 is cleaved by CI, the cleavage results in 
the formation of a short-lived binding site on the C4h fragment. A high 
density of LTA on the surface of the cell night sequester C-'ih finding 
sites or perhaps change the electrostatic charge <>f the cell surface 
sufficiently to effect the kinetics of the VAh attachment. The end 
result in either case would bn the nonprodue t i v« resumption o^ C.A 

molecules. This is consistent with the results from the residual C4 
titration studies in which no alteration of C4 consumption was observed 



i'8 



when (14 was incubated with EAC1 . This model is also copslstant with 
the Tact no dysfunction of CI, Clq, Cls, Cls, 04, or C2 coul.i he demon- 
strated when incubated fluid phase with LTApcx. 

This model would also predict that once C4b were attached to the 
membrane, subsequent addition of LTA should have significantly less 
impact on cascade disruption. As shown in Figure 17, this prediction 
coincides well with the facts. Percent inhibition of lysis drops from 
more than 65% in the case of EA treated with LTApcx (LOO ug/ml) to less 
than 20% in the case of EACH treated with the same concentration of 
LTApcx. Furthermore, EAC142 are no longer inhibited as one would 
expect if the C4b and C2a binding sites were already secured. 

Although all data thus far presented are consistant with this 
model, final proof would necessitate the 1 labelling of purified Ci 
and C2. Once labelled, one could determine if an excess of decayed C-ih 
and C2a fragments were released into the media after preincubation with 
EAC1 LTA or EAC14 respectively. 

It is hoped that future research in this area may prove enlightening 
not only in expanding upon the mechanism of inhibition but also upon 
the specific role this extracellular metabolite plays in tin- inflam- 
matory response of periodontal lesions. 

It is apparent that the anti-complementary activity of LTA is not 
restricted in a single species or geneu.s (Tabic II) and it may verv 
well be that LTA plays i s ign i I; ican t role in protecting gram positive 
organisms from Immunologic destruction. If so, LTA could be considered 
a type of "virulence" factor and those organisms (hat produce copious 
amounts of extracellular LTA (such as S. nutans BUT) would not only 
contribute to their own protection, but also to the- protection of the 



! 19 



myriad of microorganisms in their immediate environment. Obviously 
more research in this area is needed before such '-peculation can be 
substantiated with fact. 



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12-' 



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62, 



63. 



64. 



Koethe, S. M. , K. F. Austen, and I. Gigli. 1973. blocking of the 
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BIOGRAPHICAL SKETCH 

Louis (Loui) Silvestri was born in Peckville, PA on Januarv 11, 
1952. He spent most of his years in Arehbald, PA. 

Loui attended a parochial grade school (St. Thomas of Aquinas), 
a Jesuit preparatory high school (Scranton Preparatory School) and a 
college heavily influenced by Augustinian Catholicism (Villanova 
University) . 

Lout's higher education was continued at the University of Florida 
(Gainesville, PL) where, under the tutelage of Dr. Edward Hoffmann, lie 
received his Ph.D. However, earning that degree became more of a 
challenge than originally anticipated. 

Loui is currently employed at the University of Alabama (Birmingham, 
AL) as a post doctoral fellow under the direction of Dr. Robert Stroud. 



I3-J- 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 




Edward M. Hoffman^, Ichaijrman 



ogy s 



Professor of Microbiology and Cell Science 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 



- L'aCuz 




w k^xjux^ 



L. William Clem 

Professor of Immunology and Medical 

Microbiology 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 



W/ty0K> fa JgzMet. 



Brian Gebhardt' 

Associate Professor of Pathology 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 

/ ^ 



06-^^ 




Arnold S. Bleiweis 

Professor of Microbiology and Cell Science 



I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 





Vk 



L„ 0. Ingram 

Associate Professor 7 of Microbiology and 

Cell Science 



This dissertation was submitted to the Graduate Faculty of the 
Department of Microbiology and Cell Science in the College of Arts 
and Sciences and to the Graduate Council, and was accepted as 
partial fulfillment of the requirements for the degree of Doctor of 
Philosophy. 



December 1977 

Dean, Graduate School 




0* 



J 






\l